Vaccines for protecting against influenza

ABSTRACT

A vaccine containing a H1 subtype influenza A virus hemagglutinin is either unadjuvanted or is adjuvanted but not with an oil-in-water emulsion adjuvant. The vaccine is suitable for immunizing a patient against swine flu. The vaccine may be monovalent. The vaccine may include two different H1 subtype influenza A virus hemagglutinins, wherein (i) the first H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) the second H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. A monovalent vaccine may be administered in conjunction with a trivalent A/H1N1-A/H3N2 B seasonal influenza vaccine. Other embodiments are also disclosed, such as reassortant viruses.

This application claims the benefit of U.S. provisional applications61/214,787 filed Apr. 27, 2009, 61/216,198 filed May 13, 2009,61/238,628 filed Aug. 31, 2009, and 61/279,665 filed Oct. 22, 2009. Nosubject matter of these provisional applications has been inadvertentlyomitted from the present application.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002113000SEQLIST.txt,date recorded: Apr. 27, 2010, size: 52 KB).

TECHNICAL FIELD

This invention is in the field of vaccines for protecting againstinfluenza virus infection, and in particular against strains such as theswine flu strain(s) which emerged in April 2009.

BACKGROUND ART

In April 2009 a human outbreak of swine flu was confirmed in manycountries including Mexico and USA, and then spread rapidly across theglobe. A pandemic was declared by the WHO in June 2009. The disease wascaused by a newly identified swine influenza virus A/California/04/2009A(H1N1). This swine flu strain seems to have no immunologicalcross-reactivity with current human influenza vaccines strains,including the A(H1N1) antigens in current human seasonal vaccines. Thevirus has been referred to variously as ‘swine influenza’, ‘novelswine-origin H1N1 influenza’, ‘human-swine influenza’, ‘novel influenzaA(H1N1)’ and ‘influenza A(H1N1)v’.

There is a need for a vaccine to prevent further human-to-humantransmission of this swine flu and variants of it.

SUMMARY OF THE DISCLOSURE

The invention has various aspects. The invention disclosed and claimedherein does not encompass influenza vaccines having oil-in-wateremulsion adjuvants, but it does encompass of unadjuvanted influenzavaccines and vaccines having alternative adjuvants i.e. adjuvants exceptfor oil-in-water emulsions. Similarly, the invention disclosed hereindoes not encompass methods for manufacturing influenza vaccines, but itdoes encompass the vaccines themselves and their medical use. Theinvention also encompasses certain viruses.

According to a first aspect of the invention, a vaccine containing a H1subtype influenza A virus hemagglutinin is adjuvanted, but not with anoil-in-water emulsion adjuvant. The hemagglutinin elicits an immuneresponse in a recipient, and the adjuvant can enhance the heterovariantcoverage of this response. Although a particular H1 antigen might notprotect against swine flu on its own, the adjuvant can enhance theimmune response so that protection is achieved even if the vaccinehemagglutinin shows only low immunological cross-reactivity with theswine flu hemagglutinin.

Furthermore, if the vaccine includes a hemagglutinin which isimmunologically cross-reactive with the swine flu hemagglutinin thenprotection can be provided against the homologous strain and alsoagainst variants thereof, such as drift strains which can arisenaturally.

Thus the invention provides a method for immunizing a patient (typicallya human) against swine flu, comprising a step of administering to thepatient a vaccine comprising (i) a H1 subtype influenza A virushemagglutinin and (ii) an adjuvant, provided that the adjuvant is not anoil-in-water emulsion adjuvant. In some embodiments the H1 hemagglutininis more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3; in otherembodiments it is more closely related to SEQ ID NO: 3 than to SEQ IDNO: 1.

The invention provides an immunogenic composition comprising (i) a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an adjuvant, provided thatthe adjuvant is not an oil-in-water emulsion adjuvant. This compositionmay be a monovalent vaccine (i.e. it includes hemagglutinin antigen froma single influenza virus strain) but in some embodiments it may be amultivalent vaccine e.g. a trivalent vaccine also including a H3N2influenza A virus hemagglutinin and an influenza B virus hemagglutinin.

According to a second aspect of the invention, the invention provides animmunogenic composition comprising two different H1 subtype influenza Avirus hemagglutinins, wherein (i) the first H1 subtype influenza A virushemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO:3 and (ii) the second H1 subtype influenza A virus hemagglutinin is moreclosely related to SEQ ID NO: 3 than to SEQ ID NO: 1, and (iii) thecomposition is either adjuvanted or unadjuvanted, provided that when itis adjuvanted the adjuvant is not an oil-in-water emulsion adjuvant.This mixture of H1 hemagglutinins offers a broader spectrum ofprotection against H1 influenza A virus strains than currentlyavailable. This composition may also include (iii) a H3N2 and/or (iv) aninfluenza B virus antigen. In some embodiments, the composition includes(iii) a H3N2, (iv) a B/Victoria/2/87-like influenza B virus strain; and(v) a B/Yamagata/16/88-like influenza B virus strain. These compositionsmay include an immunological adjuvant, other than an oil-in-wateremulsion adjuvant.

According to a third aspect of the invention, a monovalent vaccinecontaining a H1 subtype influenza A virus hemagglutinin is administeredin conjunction with a trivalent A/H1N1-A/H3N2-B seasonal influenzavaccine, wherein the monovalent vaccine is either adjuvanted orunadjuvanted, provided that when it is adjuvanted the adjuvant is not anoil-in-water emulsion adjuvant. The monovalent vaccine includes a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 1 than to SEQ ID NO: 3; the trivalent vaccine includes a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 3 than to SEQ ID NO: 1. The monovalent vaccine may beadministered before the trivalent vaccine, after the trivalent vaccine,or at the same time. Where the two vaccines are administered separately,there may be from 2-26 weeks between the administrations. The trivalentvaccine may be adjuvanted with an oil-in-water emulsion.

In one useful embodiment, a patient first receives the trivalentseasonal vaccine (preferably adjuvanted, such as the FLUAD™ product),and later receives the monovalent vaccine (adjuvanted, but not with anoil-in-water emulsion adjuvant, or unadjuvanted). As shown herein,pre-administration of a trivalent seasonal vaccine (particularly anadjuvanted one) can improve the efficacy of a monovalent H1N1 vaccinewith a hemagglutinin more closely related to SEQ ID NO: 1 than to SEQ IDNO: 3.

In a related embodiment, a monovalent vaccine containing a H1 subtypeinfluenza A virus hemagglutinin is administered in conjunction with a4-valent A/H1N1-A/H3N2-B-B seasonal influenza vaccine, wherein the two Bstrains are a B/Victoria/2/87-like strain and a B/Yamagata/16/88-likestrain, provided that the monovalent vaccine is not adjuvanted with anoil-in-water emulsion. The monovalent vaccine includes a H1 subtypeinfluenza A virus hemagglutinin which is more closely related to SEQ IDNO: 1 than to SEQ ID NO: 3; the 4-valent vaccine includes a H1 subtypeinfluenza A virus hemagglutinin which is more closely related to SEQ IDNO: 3 than to SEQ ID NO: 1. The monovalent vaccine may be administeredbefore the trivalent vaccine, after the trivalent vaccine, or at thesame time. Where the two vaccines are administered separately, there maybe from 2-26 weeks between the administrations. The 4-valent vaccine(but not the monovalent vaccine) may e adjuvanted with an oil-in-wateremulsion. In one useful embodiment, a patient first receives themonovalent vaccine and later receives the 4-valent vaccine.

According to a fourth aspect of the invention, a monovalent vaccinecontaining a H1 subtype influenza A virus hemagglutinin is administeredby a two-dose regimen, provided that neither dose is adjuvanted with anoil-in-water emulsion (they may be unadjuvanted). The monovalent vaccineincludes a H1 subtype influenza A virus hemagglutinin which is moreclosely related to SEQ ID NO: 1 than to SEQ ID NO: 3. The two doses areadministered 1-6 weeks apart e.g. 1 week apart, 2 weeks apart, 3 weeksapart, 4 weeks apart, 5 weeks apart, 6 weeks apart. In some embodimentsthe H1 hemagglutinin is identical in both monovalent vaccines; in otherembodiments the H1 hemagglutinins in the two monovalent vaccines havedifferent amino acid sequences e.g. they may differ by up to 20 aminoacids from each other (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 amino acid substitutions).

According to a fifth aspect of the invention, an influenza A virus has agenome encoding a hemagglutinin with amino acid sequence SEQ ID NO: 6.Compared to SEQ ID NO: 1 this sequence has Pro-200 instead of Ser-200.This virus can be used with all embodiments discussed below. Theinvention also provides protein comprising amino acid sequence SEQ IDNO: 6. More generally, the invention provides an influenza A virus witha genome encoding a hemagglutinin which is more closely related to SEQID NO: 1 than to SEQ ID NO: 3, and which has a proline residue at theposition corresponding to Ser-200 in SEQ ID NO: 1. This hemagglutininmay include a HA1 sequence with at least 90% (e.g ≧91%, ≧92%, ≧93%,≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%) identity to SEQ ID NO: 2, providedthat it includes the Pro-200 residue.

According to a sixth aspect of the invention, an influenza A virus has agenome encoding a hemagglutinin with amino acid sequence SEQ ID NO: 7 orcomprising SEQ ID NO: 8. Compared to SEQ ID NO: 1 this sequence hasGlu-204 instead of Asp-204 and has a deletion of Lys-147. This virus canbe used with all embodiments discussed below. The invention alsoprovides protein comprising amino acid sequence SEQ ID NO: 7 or SEQ IDNO: 8.

The invention also provides an influenza A virus having a genomeencoding a hemagglutinin with amino acid sequence SEQ ID NO: 9 orcomprising SEQ ID NO: 10. Compared to SEQ ID NO: 7 this sequence hasSer-159 instead of Lys-159, Ser-206 instead of Gln-206, Ala-241 insteadof Glu-241, and Glu-170 instead of Lys-170. This virus can be used withall embodiments discussed below. The invention also provides proteincomprising amino acid sequence SEQ ID NO: 9 or SEQ ID NO: 10.

The invention also provides an influenza A virus having a genomeencoding a hemagglutinin with amino acid sequence SEQ ID NO: 13.Compared to SEQ ID NO: 1 this sequence has Ile-208 instead of Leu-208.This virus can be used with all embodiments discussed below. Theinvention also provides protein comprising amino acid sequence SEQ IDNO: 13. More generally, the invention provides an influenza A virus witha genome encoding a hemagglutinin which is more closely related to SEQID NO: 1 than to SEQ ID NO: 3, and which has an isoleucine residue atthe position corresponding to Leu-208 in SEQ ID NO: 1. Thishemagglutinin may include a HA1 sequence with at least 90% (e.g ≧91%,≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98%, ≧99%) identity to SEQ ID NO:2, provided that it includes the Ile-208 residue.

The invention also provides an influenza A virus with a genome encodinga hemagglutinin which is more closely related to SEQ ID NO: 1 than toSEQ ID NO: 3, and which has one or more of (i) a proline residue at theposition corresponding to Ser-200 in SEQ ID NO: 1, (ii) a glutamateresidue at the position corresponding to Asp-204 in SEQ ID NO: 1, (iii)a serine residue at the position corresponding to Lys-159 in SEQ ID NO:1, (iv) a serine residue at the position corresponding to Gln-206 in SEQID NO: 1, (v) an alanine residue at the position corresponding toGlu-241 in SEQ ID NO: 1, (vi) a glutamate residue at the positioncorresponding to Lys-170 in SEQ ID NO: 1, (vii) an isoleucine residue atthe position corresponding to Leu-208 in SEQ ID NO: 1, and/or (viii) anaspartate residue at the position corresponding to Asn-173 in SEQ IDNO: 1. These viruses can be used with all embodiments discussed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a reverse genetics experiment.

FIG. 2 shows the results of PCR amplification from rescued virus, and

FIG. 3 shows results of a restriction digest.

FIG. 4 shows H1 titers obtained after immunization with H1N1sw antigeneither unadjuvanted (0.5 or 1 μg HA dose) or adjuvanted with MF59 (0.5μg). A PBS control was also used. The black bars show titers after oneimmunization; the grey bars show titers after two immunizations.

FIG. 5 shows IgG serum antibody titers (ELISA) after two H1N1sw boostingdoses in mice primed with seasonal H1N1 (Brisbane). The priming andboosting strains and adjuvanting are indicated.

FIG. 6 shows lung viral load in ferrets immunized with variousprime/boost regimens. Animal groups A to H are described below. They-axis shows Log₁₀TCID₅₀/gr.

FIG. 7 shows nasal viral load in the same ferrets and the y-axis showslog₁₀ CDU.

FIG. 8 shows HI titers in the same ferrets.

FIGS. 9-12 show data for the F8, F9 and F10 variants obtained duringreverse genetics work, compared to A/CA/04/09 and A/PR/8/34.

FIG. 9 shows FFA titer (FFU/ml) against post-infection time (hours).

FIG. 10 shows HA titer against post-infection time.

FIG. 11 shows FFA titers for the five strains, and

FIG. 12 shows HA titers.

DETAILED DESCRIPTION OF EMBODIMENTS

Antigen components

The invention uses influenza A virus hemagglutinin as a vaccine antigen.The antigen will typically be prepared from influenza virions but, as analternative, haemagglutinin can be expressed in a recombinant host (e.g.in an insect cell line using a baculovirus vector) and used in purifiedform [1,2,3] or in the form of virus-like particles (VLPs; e.g. seereferences 4 and 5). In general, however, antigens will be from virions.

Various forms of influenza virus vaccine are currently available (e.g.see chapters 17 & 18 of reference 6). Vaccines are generally basedeither on live virus or on inactivated virus. Inactivated vaccines maybe based on whole virions, ‘split’ virions, or on purified surfaceantigens. The antigen in vaccines of the invention may take the form ofa live virus or, more preferably, an inactivated virus. Chemical meansfor inactivating a virus include treatment with an effective amount ofone or more of the following agents: detergents, formaldehyde, formalin,β-propiolactone, or UV light. Additional chemical means for inactivationinclude treatment with methylene blue, psoralen, carboxyfullerene (C60)or a combination of any thereof. Other methods of viral inactivation areknown in the art, such as for example binary ethylamine, acetylethyleneimine, or gamma irradiation.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (includinghemagglutinin and, usually, also including neuraminidase). Split virionand purified surface antigens (i.e. subvirion vaccines) are particularlyuseful with the invention.

Virions can be harvested from virus-containing fluids by variousmethods. For example, a purification process may involve zonalcentrifugation using a linear sucrose gradient solution that includesdetergent to disrupt the virions. Antigens may then be purified, afteroptional dilution, by diafiltration.

Split virions are obtained by treating virions with detergents (e.g.ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, TritonX-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9, etc.)to produce subvirion preparations, including the ‘Tween-ether’ splittingprocess. Methods of splitting influenza viruses are well known in theart e.g. see refs. 7-12, etc. Splitting of the virus is typicallycarried out by disrupting or fragmenting whole virus, whether infectiousor non-infectious with a disrupting concentration of a splitting agent.The disruption results in a full or partial solubilisation of the virusproteins, altering the integrity of the virus. Preferred splittingagents are non-ionic and ionic (e.g. cationic) surfactants e.g.alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines,betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg,alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds,sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin,lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols(e.g. the Triton surfactants, such as Triton X-100 or Triton N101),polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethyleneethers, polyoxyethlene esters, etc. One useful splitting procedure usesthe consecutive effects of sodium deoxycholate and formaldehyde, andsplitting can take place during initial virion purification (e.g. in asucrose density gradient solution). Thus a splitting process can involveclarification of the virion-containing material (to remove non-virionmaterial), concentration of the harvested virions (e.g. using anadsorption method, such as CaHPO₄ adsorption), separation of wholevirions from non-virion material, splitting of virions using a splittingagent in a density gradient centrifugation step (e.g. using a sucrosegradient that contains a splitting agent such as sodium deoxycholate),and then filtration (e.g. ultrafiltration) to remove undesiredmaterials. Split virions can usefully be resuspended in sodiumphosphate-buffered isotonic sodium chloride solution. The BEGRIVAC™,FLUARIX™, FLUZONE™ and FLUSHIELD™ products are split vaccines.

Purified surface antigen vaccines comprise the influenza surfaceantigens haemagglutinin and, typically, also neuraminidase. Processesfor preparing these proteins in purified form are well known in the art.The FLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are subunit vaccines.

Influenza antigens can also be presented in the form of virosomes [13](nucleic acid free viral-like liposomal particles), as in the INFLEXALV™ and INVAVAC™ products, but it is preferred not to use virosomes withthe present invention. Thus, in some embodiments, the influenza antigenis not in the form of a virosome.

The hemagglutinin antigen in the vaccine may be from any suitablestrain. In some embodiments the hemagglutinin is one which, whenadministered to a human subject in unadjuvanted form, elicitsanti-hemagglutinin antibodies which do not cross-react withA/California/04/2009 hemagglutinin (SEQ ID NO: 1; GI:227809830); inthese embodiments the vaccine's adjuvant enhances the immune responsesuch that a human subject produces antibodies which do cross-react withA/California/04/2009 hemagglutinin. In other embodiments thehemagglutinin is one which, when administered to a human subject inunadjuvanted form, can elicit anti-hemagglutinin antibodies which docross-react with A/California/04/2009 hemagglutinin (SEQ ID NO: 1); inthese embodiments the vaccine's adjuvant enhances the immune responsesuch that a human subject produces a broader spectrum of antibodies,which can help to protect against drift strains of A/California/04/2009.In other embodiments the hemagglutinin is from A/California/04/2009 (SEQID NO: 1). In other embodiments the hemagglutinin comprises an HA1 aminoacid sequence having at least i % sequence identity to SEQ ID NO: 2,where i is 85 or more e.g. 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 ormore (e.g. 100). Many such sequences are available e.g. from any of thefollowing known strains:

A/swine/Guangxi/17/2005, A/Swine/Ohio/891/01, A/Swine/Indiana/9K035/99,A/Swine/Indiana/P12439/00, A/swine/Minnesota/1192/2001,A/SW/MN/23124-T/01, A/swine/Guangxi/13/2006, A/swine/Minnesota/00194/2,A/SW/MN/16419/01, A/Swine/Illinois/100085A/01, A/swine/OH/511445/2007,A/Swine/Illinois/100084/01, A/Swine/North Carolina/93523/01,A/Turkey/MO/24093/99, A/swine/Korea/PZ4/2006, A/swine/Korea/PZ7/2006,A/swine/Kansas/00246/2004, A/swine/Iowa/24297/1991,A/swine/Korea/CY08/2007, A/swine/Korea/JL02/2005,A/swine/Maryland/23239/1991, A/swine/Korea/S11/2005,A/turkey/IA/21089-3/1992, A/swine/Wisconsin/1915/1988,A/Swine/Iowa/930/01, A/swine/Korea/Hongsong2/2004, A/Ohio/3559/1988,A/swine/Iowa/17672/1988, A/turkey/NC/19762/1988,A/swine/St-Hyacinthe/106/1991, A/swine/Korea/JL04/2005,A/swine/Korea/JL01/2005, A/WI/4755/1994,A/swine/California/T9001707/1991, A/swine/Korea/Asan04/2006,A/MD/12/1991, A/Swine/Wisconsin/235/97, A/swine/Kansas/3228/1987,A/Swine/Indiana/1726/1988, A/swine/Ontario/11112/04,A/Swine/Wisconsin/163/97, A/SW/MO/1877/01, A/swine/Shanghai/3/2005,A/turkey/NC/17026/1988, A/swine/Iowa/31483/1988, A/swine/Guangdong/2/01,A/swine/Iowa/1/1987, A/swine/Iowa/3/1985, A/swine/Tennessee/31/1977,etc.

Further H1N1 strains with suitable HA antigens includeA/California/04/2009 itself, A/California/7/2009, A/Texas/5/2009,A/England/195/2009, and A/New York/18/2009.

Preferred embodiments comprise a hemagglutinin which, when administeredto a human subject in unadjuvanted form, can elicit anti-hemagglutininantibodies which cross-react with A/California/04/2009 hemagglutinin(SEQ ID NO: 1), such as hemagglutinins comprising an amino acid sequencehaving at least i % sequence identity to SEQ ID NO: 2 as discussedabove.

In some embodiments, the hemagglutinin is more closely related to SEQ IDNO: 1 (A/California/04/2009) than to SEQ ID NO: 3 (A/Chile/1/1983); inother embodiments, the hemagglutinin is more closely related to SEQ IDNO: 3 than to SEQ ID NO: 1. A hemagglutinin which is more closelyrelated to SEQ ID NO: 1 than to SEQ ID NO: 3 (i.e. has a higher degreesequence identity when compared to SEQ ID NO: 1 than to SEQ ID NO: 3using the same algorithm and parameters) is referred to hereafter as a‘H1*’ hemagglutinin. SEQ ID NOs: 1 and 3 are 80.4% identical.

Useful full-length H1 hemagglutinin sequences for use with the inventioninclude SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, as well as those comprising an amino acid sequence having atleast i % sequence identity to SEQ ID NO: 2 as discussed above, orhaving at least i % sequence identity to SEQ ID NO: 12. Ideally thehemagglutinin does not include a hyper-basic regions around the HA1/HA2cleavage site. Preferred hemagglutinins have a binding preference foroligosaccharides with a Sia(α2,6)Gal terminal disaccharide compared tooligosaccharides with a Sia(α2,3)Gal terminal disaccharide (see below).

SEQ ID NO: 11 (comprising SEQ ID NO: 12) is a useful H1* hemagglutinin.It differs from SEQ ID NO: 1 at residues 214, 226 and 240 (i.e. 99.47%identity).

As well as including a H1 hemagglutinin (such as a H1* hemagglutinin),compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) additional influenza virus strains, includinginfluenza A virus and/or influenza B virus. Thus a composition mayinclude antigen from one or more strains characteristics of a normalseasonal vaccine plus at least one H1* hemagglutinin e.g. a 4-valentvaccine with two H1 strains (one a H1* hemagglutinin, one not a H1*hemagglutinin), a H3N2 strain, and one influenza B strain, or a 5-valentvaccine with two H1 strains (one a H1* hemagglutinin, one not a H1*hemagglutinin), a H3N2 strain, and two influenza B virus strains (aB/Victoria/2/87-like strain and a B/Yamagata/16/88-like strain). Theinvention also provides a 2-valent vaccine comprising a H1*hemagglutinin and a H5 hemagglutinin. Where a vaccine includes more thanone strain of influenza, the different strains are typically grownseparately and are mixed after the viruses have been harvested andantigens have been prepared. Thus a process of the invention may includethe step of mixing antigens from more than one influenza strain.

Where a vaccine of the invention includes two influenza B strains, oneB/Victoria/2/87-like strain and one B/Yamagata/16/88-like strain will beincluded. These strains are usually distinguished antigenically, butdifferences in amino acid sequences have also been described fordistinguishing the two lineages e.g. B/Yamagata/16/88-like strains often(but not always) have HA proteins with deletions at amino acid residue164, numbered relative to the ‘Lee40’ HA sequence [14]. In someembodiments of the invention where antigens are present from two or moreinfluenza B virus strains, at least two of the influenza B virus strainsmay have distinct hemagglutinins but related neuraminidases. Forinstance, they may both have a B/Victoria/2/87-like neuraminidase [15]or may both have a B/Yamagata/16/88-like neuraminidase. For instance,two B/Victoria/2/87-like neuraminidases may both have one or more of thefollowing sequence characteristics: (1) not a serine at residue 27, butpreferably a leucine; (2) not a glutamate at residue 44, but preferablya lysine; (3) not a threonine at residue 46, but preferably anisoleucine; (4) not a proline at residue 51, but at preferably a serine;(5) not an arginine at residue 65, but preferably a histidine; (6) not aglycine residue 70, but preferably a glutamate; (7) not a leucine atresidue 73, but preferably a phenylalanine; and/or (8) not a proline atresidue 88, but preferably a glutamine. Similarly, in some embodimentsthe neuraminidase may have a deletion at residue 43, or it may have athreonine; a deletion at residue 43, arising from a trinucleotidedeletion in the NA gene, has been reported as a characteristic ofB/Victoria/2/87-like strains, although recent strains have regainedThr-43 [15]. Conversely, of course, the opposite characteristics may beshared by two B/Yamagata/16/88-like neuraminidases e.g. S27, E44, T46,P51, R65, G70, L73, and/or P88. These amino acids are numbered relativeto the ‘Lee40’ neuraminidase sequence [16].

An influenza virus from which hemagglutinin protein is purified may beattenuated.

The influenza virus may be temperature-sensitive. The influenza virusmay be cold-adapted. These three features are particularly useful whenusing live virus as an antigen.

The influenza virus may be resistant to antiviral therapy (e.g.resistant to oseltamivir [17] and/or zanamivir).

In some embodiments, strains used with the invention will thus havehemagglutinin with a binding preference for oligosaccharides with aSia(α2,6)Gal terminal disaccharide compared to oligosaccharides with aSia(α2,3)Gal terminal disaccharide. Human influenza viruses bind toreceptor oligosaccharides having a Sia(α2,6)Gal terminal disaccharide(sialic acid linked α-2,6 to galactose), but eggs and Vero cells havereceptor oligosaccharides with a Sia(α2,3)Gal terminal disaccharide.Growth of human influenza viruses in cells such as MDCK providesselection pressure on hemagglutinin to maintain the native Sia(α2,6)Galbinding, unlike egg passaging. To determine if a virus has a bindingpreference for oligosaccharides with a Sia(α2,6)Gal terminaldisaccharide compared to oligosaccharides with a Sia(α2,3)Gal terminaldisaccharide, various assays can be used. For instance, reference 18describes a solid-phase enzyme-linked assay for influenza virusreceptor-binding activity which gives sensitive and quantitativemeasurements of affinity constants. Reference 19 used a solid-phaseassay in which binding of viruses to two different sialylglycoproteinswas assessed (ovomucoid, with Sia(α2,3)Gal determinants; and pigα₂-macroglobulin, which Sia(α2,6)Gal determinants), and also describesan assay in which the binding of virus was assessed against two receptoranalogs: free sialic acid (Neu5Ac) and 3′-sialyllactose(Neu5Acα2-3Galβ1-4Glc). Reference 20 reports an assay using a glycanarray which was able to clearly differentiate receptor preferences forα2,3 or α2,6 linkages. Reference 21 reports an assay based onagglutination of human erythrocytes enzymatically modified to containeither Sia(α2,6)Gal or Sia(α2,3)Gal. Depending on the type of assay, itmay be performed directly with the virus itself, or can be performedindirectly with hemagglutinin purified from the virus.

In some embodiments the H1 hemagglutinin has a different glycosylationpattern from the patterns seen in egg-derived viruses. Thus the HA (andother glycoproteins) may include glycoforms that are not seen in chickeneggs. Useful HA includes canine glycoforms.

In addition to including hemagglutinin antigen, vaccines of theinvention typically also include a neuraminidase protein e.g. thevaccine will include viral neuraminidase. The invention may protectagainst one or more of influenza A virus NA subtypes N1, N2, N3, N4, N5,N6, N7, N8 or N9, but it will usually be against N (e.g. a H1N1 virus)or N2 (e.g. a H1N2 virus). Whole virions, split virions and subunitvaccines all include both hemagglutinin and neuraminidase. When avaccine includes a neuraminidase antigen, the neuraminidase may have atleast j % sequence identity to SEQ ID NO: 4, where j is 75 or more e.g.75, 80, 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100). Manysuch sequences are available. In some embodiments, the neuraminidase ismore closely related to SEQ ID NO: 4 than to SEQ ID NO: 5. SEQ ID NOs: 4and 5 are 82% identical.

Vaccines may also include a matrix protein, such as M1 and/or M2 (or afragment thereof), and/or nucleoprotein. A pig model has shown thataddition of M2 to inactivated H1N1swine influenza virus vaccine(adjuvanted with an oil-in-water emulsion) can enhance the vaccine'sefficacy [22].

The influenza virus may be a reassortant strain, and may have beenobtained by reverse genetics techniques. Reverse genetics techniques[e.g. 23-27] allow influenza viruses with desired genome segments to beprepared in vitro using plasmids, or by plasmid-free systems. Typically,the technique involves expressing (a) DNA molecules that encode desiredviral RNA molecules e.g. from polI promoters, and (b) DNA molecules thatencode viral proteins e.g. from polII promoters, such that expression ofboth types of DNA in a cell leads to assembly of a complete intactinfectious virion. The DNA preferably provides all of the viral RNA andproteins, but it is also possible to use a helper virus to provide someof the RNA and proteins. Plasmid-based methods using separate plasmidsfor producing each viral RNA are preferred [28-30], and these methodswill also involve the use of plasmids to express all or some (e.g. justthe PB1, PB2, PA and NP proteins) of the viral proteins, with up to 12plasmids being used in some methods. If canine cells are used, a caninepolI promoter may be used [31].

To reduce the number of plasmids needed, one approach [32] combines aplurality of RNA polymerase I transcription cassettes (for viral RNAsynthesis) on the same plasmid (e.g. sequences encoding 1, 2, 3, 4, 5,6, 7 or all 8 influenza A vRNA segments), and a plurality ofprotein-coding regions with RNA polymerase II promoters on anotherplasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 8 influenzaA mRNA transcripts). The method may involve: (a) PB1, PB2 and PAmRNA-encoding regions on a single plasmid; and (b) all 8 vRNA-encodingsegments on a single plasmid. Including the NA and HA segments on oneplasmid and the six other segments on another plasmid can alsofacilitate matters.

As an alternative to using polI promoters to encode the viral RNAsegments, it is possible to use bacteriophage polymerase promoters [33].For instance, promoters for the SP6, T3 or T7 polymerases canconveniently be used. Because of the species-specificity of polIpromoters, bacteriophage polymerase promoters can be more convenient formany cell types (e.g. MDCK), although a cell must also be transfectedwith a plasmid encoding the exogenous polymerase enzyme.

In other techniques it is possible to use dual polI and polII promotersto simultaneously code for the viral RNAs and for expressible mRNAs froma single template [34,35].

An influenza A virus may include one or more RNA segments from aA/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HA and Nsegments being from a vaccine strain, i.e. a 6:2 reassortant),particularly when viruses are grown in eggs. It may also include one ormore RNA segments from a A/WSN/33 virus, or from any other virus strainuseful for generating reassortant viruses for vaccine preparation. Theinclusion of A/Ann Arbor backbone segments is also useful, particularlyfor live vaccines. Typically, the invention protects against a strainthat is capable of human-to-human transmission, and so the strain'sgenome will usually include at least one RNA segment that originated ina mammalian (e.g. in a human) influenza virus.

The viruses used as the source of the antigens can be grown either oneggs or on cell culture. The current standard method for influenza virusgrowth uses specific pathogen-free (SPF) embryonated hen eggs, withvirus being purified from the egg contents (allantoic fluid). Morerecently, however, viruses have been grown in animal cell culture and,for reasons of speed and patient allergies, this growth method ispreferred. If egg-based viral growth is used then one or more aminoacids may be introduced into the allantoid fluid of the egg togetherwith the virus [12].

When cell culture is used, the viral growth substrate will typically bea cell line of mammalian origin. Suitable mammalian cells of origininclude, but are not limited to, hamster, cattle, primate (includinghumans and monkeys) and dog cells. Various cell types may be used, suchas kidney cells, fibroblasts, retinal cells, lung cells, etc. Examplesof suitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line. Suitable dog cells are e.g.kidney cells, as in the MDCK cell line. Thus suitable cell linesinclude, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5;PER.C6; WI-38; etc.. Preferred mammalian cell lines for growinginfluenza viruses include: MDCK cells [36-39], derived from Madin Darbycanine kidney; Vero cells [40-42], derived from African green monkey(Cercopithecus aethiops) kidney; or PER.C6 cells [43], derived fromhuman embryonic retinoblasts. These cell lines are widely available e.g.from the American Type Cell Culture (ATCC) collection, from the CoriellCell Repositories, or from the European Collection of Cell Cultures(ECACC). For example, the ATCC supplies various different Vero cellsunder catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and itsupplies MDCK cells under catalog number CCL-34. PER.C6 is availablefrom the ECACC under deposit number 96022940. As a less-preferredalternative to mammalian cell lines, virus can be grown on avian celllines [e.g. refs. 44-46], including cell lines derived from ducks (e.g.duck retina) or hens. Examples of avian cell lines include avianembryonic stem cells [44,47] and duck retina cells [45]. Suitable avianembryonic stem cells, include the EBx cell line derived from chickenembryonic stem cells, EB45, EB14, and EB14-074 [48]. Chicken embryofibroblasts (CEF) may also be used.

The most preferred cell lines for growing influenza viruses are MDCKcell lines. The original MDCK cell line is available from the ATCC asCCL-34, but derivatives of this cell line may also be used. Forinstance, reference 36 discloses a MDCK cell line that was adapted forgrowth in suspension culture (‘MDCK 33016’, deposited as DSM ACC 2219).Similarly, reference 49 discloses a MDCK-derived cell line that grows insuspension in serum-free culture (‘B-702’, deposited as FERM BP-7449).Reference 50 discloses non-tumorigenic MDCK cells, including ‘MDCK-S’(ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCCPTA-6502) and ‘MDCK-SF103’ (PTA-6503). Reference 51 discloses MDCK celllines with high susceptibility to infection, including ‘MDCK.5F1’ cells(ATCC CRL-12042). Any of these MDCK cell lines can be used.

Where virus has been grown on a mammalian cell line then the compositionwill advantageously be free from egg proteins (e.g. ovalbumin andovomucoid) and from chicken DNA, thereby reducing allergenicity.

Where virus has been grown on a cell line then the culture for growth,and also the viral inoculum used to start the culture, will preferablybe free from (i.e. will have been tested for and given a negative resultfor contamination by) herpes simplex virus, respiratory syncytial virus,parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus,reoviruses, polyomaviruses, birnaviruses, circoviruses, and/orparvoviruses [52]. Absence of herpes simplex viruses is particularlypreferred.

For growth on a cell line, such as on MDCK cells, virus may be grown oncells in suspension [36, 53, 54] or in adherent culture. One suitableMDCK cell line for suspension culture is MDCK 33016 (deposited as DSMACC 2219). As an alternative, microcarrier culture can be used.

Cell lines supporting influenza virus replication are preferably grownin serum-free culture media and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentinvention in which there are no additives from serum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for viral growth. The cells growing in such culturesnaturally contain proteins themselves.

Cell lines supporting influenza virus replication are preferably grownbelow 37° C. [55] during viral replication e.g. 30-36° C., at 31-35° C.,or at 33±1° C.

The method for propagating virus in cultured cells generally includesthe steps of inoculating the cultured cells with the strain to becultured, cultivating the infected cells for a desired time period forvirus propagation, such as for example as determined by virus titer orantigen expression (e.g. between 24 and 168 hours after inoculation) andcollecting the propagated virus. The cultured cells are inoculated witha virus (measured by PFU or TCID₅₀) to cell ratio of 1:500 to 1:1,preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The virus isadded to a suspension of the cells or is applied to a monolayer of thecells, and the virus is absorbed on the cells for at least 60 minutesbut usually less than 300 minutes, preferably between 90 and 240 minutesat 25° C. to 40° C., preferably 28° C. to 37° C. The infected cellculture (e.g. monolayers) may be removed either by freeze-thawing or byenzymatic action to increase the viral content of the harvested culturesupernatants. The harvested fluids are then either inactivated or storedfrozen. Cultured cells may be infected at a multiplicity of infection(“m.o.i.”) of about 0.0001 to 10, preferably 0.002 to 5, more preferablyto 0.001 to 2. Still more preferably, the cells are infected at a m.o.iof about 0.01. Infected cells may be harvested 30 to 60 hours postinfection. Preferably, the cells are harvested 34 to 48 hours postinfection. Still more preferably, the cells are harvested 38 to 40 hourspost infection. Proteases (typically trypsin) are generally added duringcell culture to allow viral release, and the proteases can be added atany suitable stage during the culture.

Haemagglutinin (HA) is the main immunogen in inactivated influenzavaccines, and vaccine doses are standardised by reference to HA levels,typically as measured by a single radial immunodiffusion (SRID) assay.Current vaccines typically contain about 15 μg of HA per strain,although lower doses are also used e.g. for children, or in emergencysituations. Fractional doses such as 1/2 (i.e. 7.5 μg HA per strain, asin FOCETRIA™), 1/4 (i.e. 3.75 μg per strain, as in PREPANDRIX™) and 1/8have been used [56,57], as have higher doses (e.g. 3x or 9x doses[58,59]).Thus vaccines may include between 0.1 and 150 μg of HA perinfluenza strain, preferably between 0.1 and 50 μg e.g. 0.1-20 μg,0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, 3.75-15 μg etc. Particulardoses include e.g. about 45, about 30, about 15, about 10, about 7.5,about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. μg perstrain. An equal HA mass per strain is typical. Lower doses (i.e. <15μg/dose) are most useful when an adjuvant is present in the vaccine, aswith the invention. Although doses as high as 90 μg have been used insome studies (e.g. reference 60), compositions of the invention willusually include 15 μg/dose/strain or less.

HA used with the invention may be a natural HA as found in a virus, ormay have been modified.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

Compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’ e.g.polysorbate 80), an octoxynol (such as octoxynol-9 (Triton X-100) or 10,or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may include less than 1 mg/ml of each of octoxynol-10,α-tocopheryl hydrogen succinate and polysorbate 80. Other residualcomponents in trace amounts could be antibiotics (e.g. neomycin,kanamycin, polymyxin B).

Host cell DNA

Where virus has been grown on a cell line then it is standard practiceto minimize the amount of residual cell line DNA in the final vaccine,in order to minimize any oncogenic activity of the DNA. Thus thecomposition preferably contains less than 10 ng (preferably less than 1ng, and more preferably less than 100 pg) of residual host cell DNA perdose, although trace amounts of host cell DNA may be present. Ingeneral, the host cell DNA that it is desirable to exclude fromcompositions of the invention is DNA that is longer than 100 bp.

Measurement of residual host cell DNA is now a routine regulatoryrequirement for biologicals and is within the normal capabilities of theskilled person. The assay used to measure DNA will typically be avalidated assay [61,62]. The performance characteristics of a validatedassay can be described in mathematical and quantifiable terms, and itspossible sources of error will have been identified. The assay willgenerally have been tested for characteristics such as accuracy,precision, specificity. Once an assay has been calibrated (e.g. againstknown standard quantities of host cell DNA) and tested then quantitativeDNA measurements can be routinely performed. Three principle techniquesfor DNA quantification can be used: hybridization methods, such asSouthern blots or slot blots [63]; immunoassay methods, such as theThreshold™ System [64]; and quantitative PCR [65]. These methods are allfamiliar to the skilled person, although the precise characteristics ofeach method may depend on the host cell in question e.g. the choice ofprobes for hybridization, the choice of primers and/or probes foramplification, etc. The Threshold™ system from Molecular Devices is aquantitative assay for picogram levels of total DNA, and has been usedfor monitoring levels of contaminating DNA in biopharmaceuticals [64]. Atypical assay involves non-sequence-specific formation of a reactioncomplex between a biotinylated ssDNA binding protein, aurease-conjugated anti-ssDNA antibody, and DNA. All assay components areincluded in the complete Total DNA Assay Kit available from themanufacturer. Various commercial manufacturers offer quantitative PCRassays for detecting residual host cell DNA e.g. AppTec™ LaboratoryServices, BioReliance™, Althea Technologies, etc. A comparison of achemiluminescent hybridisation assay and the total DNA Threshold™ systemfor measuring host cell DNA contamination of a human viral vaccine canbe found in reference 66.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 67 & 68, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [69]while avoiding use of formaldehyde.

Vaccines containing <10 ng (e.g. <1 ng, <100 pg) host cell DNA per 15 μgof haemagglutinin are preferred, as are vaccines containing <10 ng (e.g.<1 ng, <100 pg) host cell DNA per 0.25 ml volume. Vaccines containing<10 ng (e.g. <1 ng, <100 pg) host cell DNA per 50 μg of haemagglutininare more preferred, as are vaccines containing <10 ng (e.g. <1 ng, <100pg) host cell DNA per 0.5 ml volume.

Adjuvants

Compositions of the invention can include an adjuvant which can functionto enhance the immune responses (humoral and/or cellular) elicited in apatient who receives the composition, but they do not include anoil-in-water emulsion (e.g. as in the FLUAD™ product from ChironVaccines). Instead of using oil-in-water emulsion adjuvants, otheradjuvants can also be used with the invention. Thus, for instance, anyof the following adjuvants may be used:

-   -   The adjuvants known as aluminum hydroxide and aluminum        phosphate. These names are conventional, but are used for        convenience only, as neither is a precise description of the        actual chemical compound which is present (e.g. see chapter 9 of        reference 70). The invention can use any of the “hydroxide” or        “phosphate” adjuvants that are in general use as adjuvants. The        adjuvants known as “aluminium hydroxide” are typically aluminium        oxyhydroxide salts, which are usually at least partially        crystalline. The adjuvants known as “aluminium phosphate” are        typically aluminium hydroxyphosphates, often also containing a        small amount of sulfate (i.e. aluminium hydroxyphosphate        sulfate). They may be obtained by precipitation, and the        reaction conditions and concentrations during precipitation        influence the degree of substitution of phosphate for hydroxyl        in the salt. The invention can use a mixture of both an        aluminium hydroxide and an aluminium phosphate. In this case        there may be more aluminium phosphate than hydroxide e.g. a        weight ratio of at least 2:1 e.g. ≧5:1, ≧6:1, ≧7:1, ≧8:1, ≧9:1,        etc.    -   Calcium salts, such as calcium phosphates (e.g. the “CAP”        particles disclosed in ref. 71). Nanoparticulate calcium        phosphate salts are useful e.g. with a diameter between about        300 nm and about 4000 nm.    -   Saponin formulations such as QS21 or ISCOMs (including        QS21-containing ISCOMs). ISCOMs may comprise cholesterols.    -   Non-toxic derivatives of enterobacterial lipopolysaccharide        (LPS), or lipid A derivatives. Non-toxic derivatives of LPS        include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL        (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl        lipid A with 4, 5 or 6 acylated chains. Other non-toxic LPS        derivatives include monophosphoryl lipid A mimics, such as        aminoalkyl glucosaminide phosphate derivatives e.g. RC-529        [72,73]. Lipid A derivatives include derivatives of lipid A from        Escherichia coli such as OM-174. OM-174 is described for example        in refs. 74 & 75.    -   Immunostimulatory oligonucleotides. These may contain a CpG        motif (a dinucleotide sequence containing an unmethylated        cytosine linked by a phosphate bond to a guanosine).        Double-stranded RNAs and oligonucleotides containing palindromic        or poly(dG) sequences have also been shown to be        immunostimulatory. A useful CpG adjuvant is CpG7909, also known        as ProMune™ (Coley Pharmaceutical Group, Inc.). Another is        CpG1826. As an alternative, or in addition, to using CpG        sequences, TpG sequences can be used [76], and these        oligonucleotides may be free from unmethylated CpG motifs. A        particularly useful adjuvant based around immunostimulatory        oligonucleotides is known as IC-31™ [77]. Thus an adjuvant used        with the invention may comprise a mixture of (i) an        oligonucleotide (e.g. between 15-40 nucleotides) including at        least one (and preferably multiple) CpI motifs (i.e. a cytosine        linked to an inosine to form a dinucleotide), and (ii) a        polycationic polymer, such as an oligopeptide (e.g. between 5-20        amino acids) including at least one (and preferably multiple)        Lys-Arg-Lys tripeptide sequence(s).    -   An ADP-ribosylating toxin or detoxified derivative thereof e.g.        LT-K63 or LT-R72 [78-85CT-E29H [86].    -   Chitosan.    -   Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in        diameter, more preferably ˜~200 nm to ˜30 μm in diameter, and        most preferably ˜500 nm to ˜10 μm in diameter) formed from        materials that are biodegradable and non-toxic (e.g. a        poly(α-hydroxy acid), a polyhydroxybutyric acid, a        polyorthoester, a polyanhydride, a polycaprolactone, etc.), with        poly(lactide-co-glycolide) are preferred, optionally treated to        have a negatively-charged surface (e.g. with SDS) or a        positively-charged surface (e.g. with a cationic detergent, such        as CTAB).    -   Liposomes.    -   A phosphazene, such as poly[di(carboxylatophenoxy)phosphazene]        (“PCPP”) [87,88].    -   Imidazoquinolones e.g. Imiquimod (“R-837”) [89,90], Resiquimod        (“R-848”) [91], and their analogs; and salts thereof (e.g. the        hydrochloride salts). Further details about immunostimulatory        imidazoquinolines can be found in references 92 to 96.    -   Substituted ureas [97], such as ‘ER 803058’, ‘ER 803732’, ‘ER        804053’, ER 804058’, ‘ER 804059’, ‘ER 804442’, ‘ER 804680’, ‘ER        804764’, ER 803022 or ‘ER 804057’.    -   Cyclic diguanylate (‘c-di-GMP’).    -   A thiosemicarbazone compound, such as those disclosed in        reference 98.    -   A tryptanthrin compound, such as those disclosed in reference        99.    -   A nucleoside analog, such as: (a) Isatorabine (ANA-245;        7-thia-8-oxoguanosine) and prodrugs thereof; (b) ANA975; (c)        ANA-025-1; (d) ANA380; (e) the compounds disclosed in references        100 to 102Loxoribine (7-allyl-8-oxoguanosine) [103].    -   Compounds disclosed in reference 104, including: Acylpiperazine        compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ)        compounds, Benzocyclodione compounds, Aminoazavinyl compounds,        Aminobenzimidazole quinolinone (ABIQ) compounds [105,106],        Hydrapthalamide compounds, Benzophenone compounds, Isoxazole        compounds, Sterol compounds, Quinazilinone compounds, Pyrrole        compounds [107], Anthraquinone compounds, Quinoxaline compounds,        Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole        compounds [108].    -   Compounds containing lipids linked to a phosphate-containing        acyclic backbone, such as the TLR4 antagonist E5564 [109,110]:    -   A polyoxidonium polymer [111,112] or other N-oxidized        polyethylene-piperazine derivative.    -   Methyl inosine 5′-monophosphate (“MIMP”) [113].    -   A polyhydroxlated pyrrolizidine compound [114].    -   A CD1d ligand, such as an α-glycosylceramide [115-122] (e.g.        α-galactosylceramide), phytosphingosine-containing        α-glycosylceramides, OCH, KRN7000        [(2S,3S,4R)-1-O-(α-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol],        CRONY-101, 3″-O-sulfo-galactosylceramide, etc.    -   An inulin, such as a gamma inulin [123] or derivative thereof,        such as algammulin.        Pharmaceutical compositions

Compositions of the invention are pharmaceutically acceptable. Theyusually include components in addition to the antigens e.g. theytypically include one or more pharmaceutical carrier(s) and/orexcipient(s). A thorough discussion of such components is available inreference 124.

Compositions will generally be in aqueous form.

The composition may include preservatives such as thiomersal (e.g at 10μg/ml) or 2-phenoxyethanol. It is preferred, however, that the vaccineshould be substantially free from (i.e. less than 5 μg/ml) mercurialmaterial e.g. thiomersal-free [125]. Vaccines containing no mercury aremore preferred. Preservative-free vaccines are particularly preferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Compositions will generally have an osmolality of between 200 mOsm/kgand 400 mOsm/kg, preferably between 240-360 mOsm/kg, and will morepreferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [126], but keeping osmolality in this range is neverthelesspreferred.

Compositions may include one or more buffers. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer; or a citrate buffer. Buffers will typically beincluded in the 5-20 mM range. The buffer may be in an emulsion'saqueous phase.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.A process of the invention may therefore include a step of adjusting thepH of the bulk vaccine prior to packaging.

The composition is preferably sterile. The composition is preferablygluten free.

Preferred vaccines have a low endotoxin content e.g. less than 1 IU/ml,and preferably less than 0.5 IU/ml. The international unit for endotoxinmeasurement is well known and can be calculated for a sample by, forinstance, comparison to an international standard [127,128], such as the2nd International Standard (Code 94/580-IS) available from the NIBSC.Current vaccines prepared from virus grown in eggs have endotoxin levelsin the region of 0.5-5 IU/ml.

The vaccine is preferably free from antibiotics (e.g. neomycin,kanamycin, polymyxin B).

The composition may include material for a single immunisation, or mayinclude material for multiple immunisations (i.e. a ‘multidose’composition). Multidose arrangements usually include a preservative inthe vaccine. To avoid this need, a vaccine may be contained in acontainer having an aseptic adaptor for removal of material.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children, and unit doses will be selected accordinglye.g. a unit dose to give a 0.5 ml dose for administration to a patient.

Packaging of compositions or kit components

Processes of the invention can include a step in which vaccine is placedinto a container, and in particular into a container for distributionfor use by physicians.

Suitable containers for the vaccines include vials, nasal sprays anddisposable syringes, which should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colorless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial, and the contents of the vial can be removed backinto the syringe. After removal of the syringe from the vial, a needlecan then be attached and the composition can be administered to apatient. The cap is preferably located inside a seal or cover, such thatthe seal or cover has to be removed before the cap can be accessed. Avial may have a cap that permits aseptic removal of its contents,particularly for multidose vials.

Where a composition/component is packaged into a syringe, the syringemay have a needle attached to it. If a needle is not attached, aseparate needle may be supplied with the syringe for assembly and use.Such a needle may be sheathed. Safety needles are preferred. 1-inch23-gauge, 1-inch 25-gauge and ⅝-inch 25-gauge needles are typical.Syringes may be provided with peel-off labels on which the lot number,influenza season and expiration date of the contents may be printed, tofacilitate record keeping. The plunger in the syringe preferably has astopper to prevent the plunger from being accidentally removed duringaspiration. The syringes may have a latex rubber cap and/or plunger.Disposable syringes contain a single dose of vaccine. The syringe willgenerally have a tip cap to seal the tip prior to attachment of aneedle, and the tip cap is preferably made of a butyl rubber. If thesyringe and needle are packaged separately then the needle is preferablyfitted with a butyl rubber shield. Preferred syringes are those marketedunder the trade name “Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A composition may be combined (e.g. in the same box) with a leafletincluding details of the vaccine e.g. instructions for administration,details of the antigens within the vaccine, etc. The instructions mayalso contain warnings e.g. to keep a solution of adrenaline readilyavailable in case of anaphylactic reaction following vaccination, etc.

Methods of treatment, and administration of the vaccine

Compositions of the invention are suitable for administration to humanpatients, and the invention provides a method of raising an immuneresponse in a patient, comprising the step of administering acomposition of the invention to the patient.

The invention also provides a kit or composition of the invention foruse as a medicament.

The immune response raised by the methods and uses of the invention willgenerally include an antibody response, preferably a protective antibodyresponse. Methods for assessing antibody responses, neutralisingcapability and protection after influenza virus vaccination are wellknown in the art. Human studies have shown that antibody titers againsthemagglutinin of human influenza virus are correlated with protection (aserum sample hemagglutination-inhibition titer of about 30-40 givesaround 50% protection from infection by a homologous virus) [129].Antibody responses are typically measured by hemagglutinationinhibition, by microneutralisation, by single radial immunodiffusion(SRID), and/or by single radial hemolysis (SRH). These assay techniquesare well known in the art.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal [130-132], intradermal [133,134], oral [135],transcutaneous, transdermal [136], etc. Intradermal and intranasalroutes are attractive. Intradermal administration may involve amicroinjection device e.g. with a needle about 1.5 mm long.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusthe patient may be less than 1 year old (e.g. <6 months old), 1-5 yearsold, 5-15 years old, 15-55 years old, or at least 55 years old.Preferred patients for receiving the vaccines are the elderly (e.g. ≧50years old, ≧60 years old, and preferably ≧65 years), the young (e.g. ≦5years old, or those aged between 6 months and 24 years, or between 6months and 4 years, or between 5-18 years), middle aged (25-64 yearsold), hospitalised patients, healthcare workers, armed service andmilitary personnel, pregnant women, the chronically ill, immunodeficientpatients, patients who have taken an antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population.

Some older adults (about a third of those older than 60 years) but fewyoung adults and essentially no children have pre-existing serumantibody against the pandemic A/CA/04/09 strain. Seasonal immunizationof young people does not elicit antibodies against this strain [137]. Auseful group of subjects to receive immunogenic compositions of theinvention, particularly such compositions comprising an oil-in-wateradjuvant, is those subjects who have no existing serum antibody againstthe pandemic A/CA/04/09 strain e.g. patients born after 1960, after1970, after 1980, after 1990, or after 2000.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are: (1)≧70% seroprotection; (2) ≧40% seroconversion; and/or (3) a GMT increaseof ≧2.5-fold. In elderly (>60 years), these criteria are: (1) ≧60%seroprotection; (2) ≧30% seroconversion; and/or (3) a GMT increase of≧2-fold. These criteria are based on open label studies with at least 50patients. The criteria apply for each strain in a vaccine.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients e.g. for peoplewho have never received an influenza vaccine before, or for vaccinatingagainst a new HA subtype. Multiple doses will typically be administeredat least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks,about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H.influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a respiratory syncytial virus vaccine, a pneumococcalconjugate vaccine, etc. Administration at substantially the same time asa pneumococcal vaccine and/or a meningococcal vaccine is particularlyuseful in elderly patients.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™). Another antiviral which can be administered is thymosinalpha 1 (e.g. thymalfasin, a 28 amino acid synthetic peptide, availableas ZADAXIN™), particularly when used in combination with a vaccine whichincludes an oil-in-water emulsion adjuvant [138]. In one specificembodiment, a patient receives a neuraminidase inhibitor, such asoseltamivir phosphate, at substantially the same time as receiving aninactivated whole virion vaccine (e.g. monovalent, H1*).

Vaccine products and kits

The invention also provides an unadjuvanted vaccine comprising a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 1 than to SEQ ID NO: 3. This composition is a monovalentinactivated surface antigen vaccine. The inactivated viruses may havebeen grown on eggs or in MDCK cell culture. The vaccine has a unit doseincluding about 15 μg of the H1 hemagglutinin (e.g. aA/California/7/2009-like strain, such as from reassortant strain X179A).The vaccine is unadjuvanted. It may be administered intramuscularly e.g.to the deltoid or anterolateral thigh. A subject may receive a singledose of the vaccine or may receive two doses (e.g. separated by between2 weeks and 6 months e.g. 3 weeks apart).

The invention also provides a vaccine comprising (i) a H1 subtypeinfluenza A virus hemagglutinin which is more closely related to SEQ IDNO: 1 than to SEQ ID NO: 3 and (ii) an adjuvant comprising bothaluminium phosphate and aluminium hydroxide. In a useful embodiment,this composition is a monovalent inactivated whole virion vaccine. Theinactivated viruses may have been grown on eggs. The vaccine may bepresented in a syringe containing a 0.5 ml unit dose, with each unitdose including about 15 μg of the H1 hemagglutinin. The adjuvant mayinclude, in a 0.5 ml volume, about 0.5 mg of Al⁺⁺⁺ e.g. 0.45 mg fromaluminium phosphate and 0.05 mg from aluminium hydroxide, hydrated. Thecomposition may include 50 μg of thiomersal.

The invention also provides a vaccine comprising (i) a H1 subtypeinfluenza A virus hemagglutinin which is more closely related to SEQ IDNO: 1 than to SEQ ID NO: 3 and (ii) an aluminium phosphate adjuvant. Ina useful embodiment, this composition is a monovalent inactivated wholevirion vaccine (e.g. obtained by formaldehyde inactivation). Theinactivated viruses may have been grown on eggs. The vaccine may bepresented in an ampoule containing a 0.5 ml unit dose, with each unitdose including about 6 μg of the H1 hemagglutinin (e.g. from areassortant strain, such as X-179A). The hemagglutinin may be adsorbedto the aluminium phosphate adjuvant, which may be in the form of a gel.The composition may include thiomersal.

The invention also provides an unadjuvanted vaccine comprising a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 1 than to SEQ ID NO: 3 (e.g. from a A/California/7/2009strain). In a useful embodiment, this composition is a monovalentinactivated whole virion vaccine in which the inactivated viruses weregrown on Vero cells. The vaccine may be presented in a vial containingmultiple doses e.g. 10×0.5 ml unit dose, with each unit dose includingabout 7.5 μg of the H1 hemagglutinin (i.e. 75 μg per multidose vial).The composition may include trometamol, sodium chloride and polysorbate80. The composition may include Tris-buffered saline and polysorbate 80.The vaccine may be administered intramuscularly e.g. to the deltoid oranterolateral thigh. The vaccine may be administered as two doses, givenat least 3 weeks apart.

The invention also provides an unadjuvanted vaccine comprising a H1subtype influenza A virus hemagglutinin which is more closely related toSEQ ID NO: 1 than to SEQ ID NO: 3 (e.g. from a A/California/7/2009strain, such as X-179A). In a useful embodiment, this composition is amonovalent split virion inactivated vaccine in which the inactivatedviruses were grown in eggs. The vaccine may be presented in a vialcontaining multiple doses (e.g. 10×0.5 ml unit dose with a thimerosalpreservative, which may be present at 45 μg per 0.5 ml unit dose) or insyringes (one dose per syringe). The vaccine may be administeredintramuscularly e.g. to the deltoid or anterolateral thigh. Each dose ofvaccine may have 7.5, 15 or 30 μg of the H1 hemagglutinin. The vaccinemay include a phosphate buffer.

The invention also provides a kit comprising (i) a first kit componentcomprising influenza A virus hemagglutinin, but not including a H1*hemagglutinin and (ii) a second kit component comprising an influenza Avirus H1* hemagglutinin, provided that the second kit component does notcomprise an oil-in-water emulsion adjuvant. Mixture of the two kitcomponents gives a combined vaccine for administration to a subject. Thesecond kit component is preferably monovalent i.e. it includes only onetype of influenza hemagglutinin, namely the H1* hemagglutinin. The firstkit component can be, for example: (a) a 3-valent vaccine compositionincluding antigen from a H1N1 strain, a H3N2 strain, and one influenza Bstrain; (b) a 4-valent vaccine composition including antigen from a H1N1strain, a H3N2 strain, a B/Victoria/2/87-like influenza B virus strainand a B/Yamagata/16/88-like influenza B virus strain; or (c) amonovalent vaccine composition comprising antigen from a H5N1 influenzaA virus strain. In some embodiments, the first kit component includes anadjuvant, such as an oil-in-water emulsion.

The invention also provides a method for preparing an influenza vaccine,comprising a step of mixing a first kit component as defined in thepreceding paragraph with a second kit component as defined in thepreceding paragraph.

Vaccines mentioned in this section can usefully include a hemagglutinincomprising SEQ ID NO: 12.

Reassortant viruses

As mentioned above, the invention can use a reassortant influenza virusstrain. Thus the invention provides an influenza A virus, wherein (a)the viral hemagglutinin gene encodes a hemagglutinin which is moreclosely related to SEQ ID NO: 1 than to SEQ ID NO: 3, and (b) at leastone other viral gene is from the A/PR/8/34 influenza virus strain(A/Puerto Rico/8/34). Thus the virus may include at least one ofsegments NP, M, NS, PA, PB1 and/or PB2 from A/PR/8/34.

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin which is more closely relatedto SEQ ID NO: 1 than to SEQ ID NO: 3 and (b) at least one other viralgene is from the AA/6/60 influenza virus strain (A/Ann Arbor/6/60). Thusthe virus may include at least one of segments NP, M, NS, PA, PB1 and/orPB2 from AA/6/60. The AA/6/60 strain may be a cold-adapted AA/6/60strain e.g. its PB1 may include one or more of K391E, E581G &/or A661Tmutations in PB1, a N265S mutation in PB2, and/or a D34G mutation in NP[139].

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin which is more closely relatedto SEQ ID NO: 1 than to SEQ ID NO: 3, and (b) at least one other viralgene is from the A/WSN/33 influenza virus strain. Thus the virus mayinclude at least one of segments NP, M, NS, PA, PB1 and/or PB2 fromA/WSN/33.

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin protein which has at least k% sequence identity to SEQ ID NO: 1, where k is 85 or more e.g. 85, 88,90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100), and (b) at least oneother viral gene is from the A/PR/8/34 influenza virus strain (A/PuertoRico/8/34). Thus the virus may include at least one of segments NP, M,NS, PA, PB1 and/or PB2 from A/PR/8/34.

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin protein which has at least k% sequence identity to SEQ ID NO: 1, where k is 85 or more e.g. 85, 88,90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100), and (b) at least oneother viral gene is from the AA/6/60 influenza virus strain (A/AnnArbor/6/60). Thus the virus may include at least one of segments NP, M,NS, PA, PB1 and/or PB2 from AA/6/60.

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin protein which has at least k% sequence identity to SEQ ID NO: 1, where k is 85 or more e.g. 85, 88,90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100), and (b) at least oneother viral gene is from the A/WSN/33 influenza virus strain. Thus thevirus may include at least one of segments NP, M, NS, PA, PB1 and/or PB2from A/WSN/33.

In these viruses, the viral neuraminidase gene may encode aneuraminidase protein which has at least j % sequence identity to SEQ IDNO: 4, where j is 75 or more e.g. 75, 80, 85, 88, 90, 92, 94, 95, 96,97, 98, 99 or more (e.g. 100). In some embodiments, the neuraminidase ismore closely related to SEQ ID NO: 4 than to SEQ ID NO: 5.

The eight segments of the influenza A virus genome encode (i) the PAsubunit of the viral polymerase (ii) the PB1 subunit of the viralpolymerase (iii) the PB2 subunit of the viral polymerase (iv) the viralnucleoprotein (v) the viral matrix proteins (vi) the viral NS1 and NS2proteins (vii) hemagglutinin and (viii) neuraminidase. Preferredreassortants of the invention are 6:2 reassortants i.e. they include 6segments from one strain (e.g. from A/PR/8/34, A/WSN/33 or AA/6/60) butthe HA and NA segments from a different strain (e.g. as defined above byreference to SEQ ID NOs 1 and 4). In other embodiments there is a 7:1reassortant with HA as defined above. In other embodiments the virusincludes genes with three different origins, but with at least onesegment (e.g. 1, 2, 3, 4, 5, 6) being from A/PR/8/34, A/WSN/33 and/orAA/6/60.

Viral segments from the A/PR/8/34, A/WSN/33 and AA/6/60 strains arewidely available. Their sequences are available on the public databasese.g. GI:89779337, GI:89779334, GI:89779332, GI:89779320, GI:89779327,GI:89779325, GI:89779322, GI:89779329.

Reassortant viruses of the invention may have a H1* hemagglutinin with abinding preference for oligosaccharides with a Sia(α2,6)Gal terminaldisaccharide compared to oligosaccharides with a Sia(α2,3)Gal terminaldisaccharide.

A reassortant virus of the invention may have amino acid proline atresidue 200 (numbered according to SEQ ID NO: 1). For example, it mayencode hemagglutinin having sequence SEQ ID NO: 6. Other reassortantsmay encode hemagglutinin having sequence SEQ ID NO: 7 or SEQ ID NO: 9.

These viruses are particularly useful for preparing vaccines and canconveniently be prepared by reverse genetics. Vaccines including one ormore A/PR/8/34 or A/WSN/33 viral genes may be used to prepareinactivated influenza vaccines. Vaccines including one or more AA/6/60viral genes may be used to prepare live attenuated influenza vaccines.

The reassortant viruses of the invention can grow in MDCK cells, and theinvention provides a method of preparing a virus, comprising steps of:(i) infecting a cell culture with a virus of the invention; (ii)culturing the cell culture from step (i) to produce further virus; and(iii) purifying virus obtained in step (ii). The cell culture in step(i) is preferably a MDCK cell culture, but other cells (ideallymammalian cells, such as PER.C6 cells) may be used as an alternative.

The invention also provides a host cell comprising one or moreexpression construct(s) for providing said reassortant strains. Thus theconstruct(s) may encode a viral hemagglutinin gene with a hemagglutininwhich is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3. Theconstruct(s) will additionally encode the other viral segments for thefunctional influenza genome, such as (i) at least one viral segment fromthe A/PR/8/34 influenza virus strain; (ii) at least one other viralsegment from the AA/6/60 influenza virus strain; (iii) at least oneother viral segment from the A/WSN/33 influenza virus strain; etc. Theneuraminidase segment may encode a neuraminidase protein which has atleast j % sequence identity to SEQ ID NO: 4, etc.

The invention also provides a construct or set of constructs encodingthese reassortant strains e.g. when introduced into a host cell. Use ofthe construct(s) will provide an infectious influenza virus in asuitable reverse genetics host system. The constructs may be plasmids ornon-plasmid vectors.

The invention also provides a process for RNA expression in a host cell,comprising the use of such construct(s). The invention also provides amethod for producing a reassortant virus from such construct(s) and/orhost cell(s).

NS1 mutant viruses

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin which is more closely relatedto SEQ ID NO: 1 than to SEQ ID NO: 3, and (b) the viral genome does notencode a NS1 protein. The invention also provides an influenza A virus,wherein (a) the viral hemagglutinin gene encodes a hemagglutinin proteinwhich has at least k % sequence identity to SEQ ID NO: 1, where k is 85or more e.g. 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100),and (b) the viral genome does not encode a NS1 protein. NS1 knockoutmutants are described in reference 140.

The invention also provides an influenza A virus, wherein (a) the viralhemagglutinin gene encodes a hemagglutinin which is more closely relatedto SEQ ID NO: 1 than to SEQ ID NO: 3, and (b) the viral genome encodes atruncated NS1 protein. The invention also provides an influenza A virus,wherein (a) the viral hemagglutinin gene encodes a hemagglutinin proteinwhich has at least k % sequence identity to SEQ ID NO: 1, where k is 85or more e.g. 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100),and (b) the viral genome encodes a truncated NS1 protein. Suitabletruncations are known in the art (e.g. see references 141 & 142) andinclude truncations which leave only the first N-terminal 126 aminoacids of NS1.

These NS1 -mutant virus strains are particularly suitable for preparinglive attenuated vaccines.

The NS1 -mutant viruses ideally express PA, PB1, PB2, nucleoprotein,matrix, hemagglutinin and neuraminidase proteins.

Combination vaccines

In addition to the strain and vaccine combinations discussed above, theinvention provides a multivalent immunogenic composition comprising (i)a H1 subtype influenza A virus hemagglutinin which is more closelyrelated to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an influenza Avirus hemagglutinin from 1, 2, 3 or 4 of hemagglutinin subtypes H2, H5,H7 and/or H9. Thus the composition may be H1-H2 bivalent, H1-H7bivalent, H1-H2-H5 trivalent, H1-H5-H7-H9 tetravalent, H1-H2-H5-H7-H9pentavalent, etc. At least two strains in the vaccine may share a commonneuraminidase subtype e.g. a H1N1-H2N1 bivalent, H1N1-H2N2-H5N1trivalent, etc.

Antibodies

The invention provides a monoclonal antibody which can bind to SEQ IDNO: 1 with higher affinity than to SEQ ID NO: 3. These monoclonalantibodies can be used in therapy.

Immunoassays

The invention provides an immunoassay for an influenza vaccine (e.g. aSRID assay) in which porcine anti-hemagglutinin antibodies are employed.These antibodies may be obtained from a pig following a swine fluinfection. For instance, archived samples of swine sera may be used as asource of antibodies, thereby avoiding the delay typical for developmentof SRID reagents.

The invention also provides a gel including a porcine antiserumcontaining antibodies that recognize influenza hemagglutinin, and inparticular which recognize the hemagglutinin of an influenza virus whichcan infect humans e.g. A/California/04/2009. The gel is suitable forperforming a SRID assay e.g. it is an agarose gel.

The invention also provides an immunoassay for an influenza vaccine(e.g. a SRID assay) in which anti-hemagglutinin antibodies are employed,wherein the antibodies recognize the hemagglutinin of a swine influenzavirus isolated in a year before 2009. The invention also provides a gelincluding an antiserum containing such antibodies. The gel is suitablefor performing a SRID assay e.g. it is an agarose gel.

In some embodiments of the invention, HA concentration is not measuredby SRID or by any other immunoassay, but is instead measured by analternative assay e.g. by a chromatographic technique, such as byreverse phase high performance liquid chromatography (RP-HPLC).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

“GI” numbering is used above. A GI number, or “GenInfo Identifier”, is aseries of digits assigned consecutively to each sequence recordprocessed by NCBI when sequences are added to its databases. The GInumber bears no resemblance to the accession number of the sequencerecord. When a sequence is updated (e.g. for correction, or to add moreannotation or information) then it receives a new GI number. Thus thesequence associated with a given GI number is never changed.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

Where a cell substrate is used for reassortment or reverse geneticsprocedures, it is preferably one that has been approved for use in humanvaccine production e.g. as in Ph Eur general chapter 5.2.3.

Identity between polypeptide sequences is preferably determined by theSmith-Waterman homology search algorithm as implemented in the MPSRCHprogram (Oxford Molecular), using an affine gap search with parametersgap open penalty=12 and gap extension penalty=1.

MODES FOR CARRYING OUT THE INVENTION

Prime/boost regimen

A single dose of adjuvanted seasonal FLUAD™ vaccine is administered tosubjects. Several weeks later (from 2-26 weeks later) they receive anunadjuvanted monovalent vaccine including hemagglutinin fromA/California/04/2009 (SEQ ID NO: 1).

Reassortant strain

Mammalian cells were transfected with all 6 backbone segments ofinfluenza A virus strain A/PR/8/34 and the two surface glycoproteinsegments from an A/California/04/09 H1N1 strain in a reverse geneticssystem. An initial assay of culture fluid recovered from the transfectedcells showed five positive results for rescued virus (FIG. 1A) andpassaging of this material gave many more positive results (FIG. 1B).

To confirm that the observed viruses were true reassortants,strain-specific PCR primers were used to detect the HA, NA and PB1genes. As shown in FIG. 2, the rescued virus (lanes ‘1’) and the plasmidDNA used in the reverse genetics system (lanes ‘2’) had the same sizefor all three genes, whereas PCR performed on water (lanes ‘3’) showedno amplification.

A restriction digest was then performed on the PCR products. The PB1gene in PR/8/34 includes a SalI site, whereas the PB1 gene in A/CA/04/09does not. The HA gene in A/CA/04/09 includes a KpnI site, whereas the HAgene in PR/8/34 does not. The NA gene in A/CA/04/09 includes aEcoRVsite, whereas the NA gene in PR/8/34 does not. Thus these threerestriction enzymes can distinguish between the two strains. As shown inFIG. 3 the PCR products (lanes ‘A’) were digested in all three cases,and the digestion products for the rescued reassortant (lanes ‘B’) andthe original plasmid DNA (lanes ‘C’) were identical. Thus the virusproduced by the reverse genetics system included the HA and NA genesfrom A/CA/04/09 and PB1 from A/PR/8/34, showing that it has beenpossible to produce an infectious reassortant virus.

Ferret study I

Reference 143 reports a ferret model for investigating influenzavaccines. Ferrets were primed with an adjuvanted (squalene-containingoil-in-water emulsion, MF59™; not an embodiment of the invention) orunadjuvanted seasonal vaccine, or with PBS. Three weeks later (day 21)these ferrets received a booster dose of adjuvanted or unadjuvantedtrivalent seasonal or a monovalent pandemic (‘H1N1sw’) vaccine, or PBS.Eight animal groups A to H were used in total:

Group A B C D K F G H Prime S,A S,A S,A S Ss S PBS PBS Boost S,A sw sw,AS sw sw,A sw,A PBS S = seasonal, sw = swine, A = adjuvanted

At day 49 ferrets were then challenged with a H1N1sw strain (10⁶ TCID₅₀)and lung pathology was assessed in each group. Unlike seasonal H1N1,which infects only nose and trachea, the H1N1sw virus also infects thelungs. The H1N1sw virus is not lethal for the ferrets.

The average % of affected lung parenchyma were:

Group A B C D E F G H % 13.3 6.7 3.3 15.8 7.5 6.7 3 3 48.3

Thus, compared to the PBS group, all ferrets previously primed witheither adjuvanted or unadjuvanted seasonal vaccine and then boosted witheither the homologous seasonal or with H1N1sw had an important reductionin the lung pathology.

Lung viral load was also assessed and results are shown in FIG. 6. Ascompared to PBS, one dose of adjuvanted H1N1sw vaccine reduced lungviral load by 2 to 3 logs (compare groups G & H). The viral load in thelungs was reduced to almost undetectable levels (group F) if the H1N1swvaccination was preceded by administration of an unadjuvanted seasonalinfluenza vaccine.

Viral load was also assessed from nasal swabs (FIG. 7). Similar resultswere found in throat swabs.

HI antibody responses were also measured at day 49 (FIG. 8). One dose ofadjuvanted H1N1sw vaccine was more immunogenic than unadjuvanted H1N1swvaccine. HI titers against H1N1sw virus increased by at least 1 log inferrets previously immunized with adjuvanted seasonal vaccine.

HI antibody responses against seasonal H1N1 and H3N2 seasonal strainswere also assessed. Antibodies which cross-react between seasonal H1N1and H1N1sw were not detected by HI.

Thus one dose of adjuvanted H1N1sw vaccine was more immunogenic and moreefficacious than unadjuvanted vaccine, measured by viral loads in lungs,nose, and throat. Both immunogenicity and efficacy were enhanced byprevious immunization with seasonal influenza vaccine, and this effectwas better if the seasonal vaccine was adjuvanted. This enhancement ofimmunogenicity and efficacy does not appear to be due to antibodiescross-reacting (by HI) between seasonal H1N1 and H1N1sw.

These results can explain why elderly people might be better protectedagainst H1N1sw virus despite little cross-reactivity of antibodies. Theycan also explain the preliminary results of clinical trials showing goodresponse after one single dose in healthy adults, as this effect couldbe due to previous immunological experience with seasonal viruses (vianatural infection or vaccination), despite little or no cross-reactivityof antibodies. The results also imply that better H1N1sw protection isachieved in the presence of an adjuvant and if a patient has previouslybeen immunized with adjuvanted seasonal vaccine. The data suggest thatimmunologically naive individuals (e.g. children) and immunologicallyfrail individuals may require more than one dose of adjuvanted H1N1swvaccine for optimal and sustained protection even though a single dosecan still be clinically useful.

Further details of this ferret study are in reference 144.

Mouse study I

In unprimed mice, without any prior exposures to flu antigens, a singledose of emulsion-adjuvanted H1N1sw vaccine (not an embodiment of theinvention) gives HI titers associated with protection in humans. Withoutthe adjuvant, two doses were required to reach this titer.

Vaccines were prepared from H1N1sw A/California/07/2009 H1N1-likeviruses grown in eggs. Vaccines were either unadjuvanted or wereadjuvanted with an oil-in-water emulsion comprising squalene (MF59™).Vaccines were standardized by SRID with a HA dose of either 0.5 μg or 1μg. Balb/c mice aged 6-7 weeks were immunized intramuscularly on day 0with phosphate buffered saline, with 0.5 or 1.0 μg (HA content) ofantigen alone, or with 0.5 μg of antigen with 50 μl of adjuvant. Dosevolume was 100 μl. Sera were obtained on day 13. Mice were boosted witha second dose, matching the first, on day 14. Sera were again collectedon day 21. Sera were assayed by hemagglutination inhibition (HI) usinginactivated whole virus for antigen and turkey red blood cells.

A single immunization with 0.5 μg adjuvanted antigen elicited an averagefunctional antibody (HI) titer of 1:63 in serum obtained two weeks afterimmunization (FIG. 4). A HI titer of 1:40 or more is associated withprotection of humans from seasonal influenza [145]. A secondimmunization with adjuvanted vaccine two weeks later increased theaverage HI titer to 1:1280 in serum obtained one week after the boost. Asingle immunization with antigen without adjuvant did not elicitsignificant HI titers, but a second immunization two weeks laterelicited a HI titer of 1:160. There was no significant difference intiters elicited by immunization with 0.5 or 1.0 μg of unadjuvantedantigen.

These data are consistent with results of human immunization withvaccines against other potential pandemic influenza strains. Withoutadjuvant, vaccines against H5 avian influenza strains elicit lowantibody titers; MF59 greatly increases the rapidity, titer, and breadthof the elicited antibodies [146,147]. During a much smaller humanoutbreak of swine origin influenza in 1976, adjuvanted vaccines were notavailable. A single dose of the 1976 vaccines elicited low antibodytiters in young people, but significantly higher titers in olderindividuals, probably because older subjects had experienced morepriming influenza infections or immunizations [148].

Mouse study II

Mice primed with seasonal H1N1 (A/Brisbane/59/2007; 0.2 μg HA dose)monovalent vaccine (with or without MF59 adjuvant) were boosted twice(days 36 and 66) with the same vaccine or with equivalent monovalentvaccines (again, with or without MF59 adjuvant) prepared from pandemicH1N1sw strains (A/California/04/2009 hemagglutinins).

ELISA analysis of the immune responses (FIG. 5) suggests that priorseasonal adjuvanted vaccination effectively primed the mice for a highertiter response to the H1N1sw vaccine, and this priming was especiallyimportant if the H1N1sw vaccine was unadjuvanted. In unprimed mice ormice primed with unadjuvanted seasonal H1N1, a high titer response wasseen only if the H1N1sw vaccine was adjuvanted.

Thus adjuvanting of the H1N1sw vaccine seems to be important for arobust immune response. Moreover, adjuvanting seems to be important forallowing the seasonal vaccine to prime for a robust antibody response tothe pandemic vaccine.

In summary, immunization with two doses of unadjuvanted pandemic vaccineelicited little functional antibody in un-primed mice or in mice primedwith unadjuvanted seasonal vaccine. In mice primed with adjuvantedseasonal vaccine, however, two doses of unadjuvanted pandemic vaccinegave a good response. Mice responded robustly to two doses of adjuvantedpandemic vaccine regardless of whether they had been primed. Althoughadjuvanted seasonal vaccines may not efficiently elicit antibodiesagainst the pandemic strain, therefore, they may prime for a highertiter response to pandemic vaccines.

Mouse study III

Three groups of 40 6-week-old female BALB/c mice received a single i.m.injection of a trivalent seasonal vaccine, from either the 2005/06season or the 2009/10 season (both northern hemisphere). Influenza-naivecontrol mice received PBS. The vaccines were administered at 1/10th thehuman dose (1.5 μg HA per strain) on day 0. On day 40 mice were dividedinto four subgroups of 10 animals each and were re-vaccinated with amonovalent inactivated H1N1sw vaccine. The four groups received a highor low dose (3 μg HA or 0.3 μg HA), with or without a submicronoil-in-water emulsion adjuvant comprising squalene in combination withsorbitan oleate, polyoxyethylene cetostearyl ether and mannitol. Allanimals then received a second H1N1sw dose at day 61. The presence of HIantibodies against the seasonal and pandemic H1N1 strains was assessedat days 40, 61, 75 and 102. Full details of this mouse study are givenin reference 149.

The results confirmed that a single injection of the H1N1sw vaccine wassufficient to induce HI antibody responses to protective levels, with orwithout adjuvant. The HI antibody titer (GMT) against the H1N1sw strainwas >40 in all groups except for the group of naïve mice immunized with0.3 μg HA of unadjuvanted vaccine.

Antibodies elicited by previous seasonal influenza vaccination did notcross-react with the H1N1sw strain, but priming with seasonal influenzavaccines did result in higher antibody responses to non-adjuvantedH1N1sw vaccine. In contrast, previous seasonal immunization did notappear to influence the immunogenicity of the adjuvanted H1N1sw vaccinein mice, likely due to a strong primary response induced by theadjuvanted vaccine in these groups.

Mouse study IV

VLPs were prepared from recombinant H1N1sw HA expressed in Sf9 insectcells. The VLPs contained ˜0.1 mg HA per mg of total VLP protein. FemaleBALB/c mice were immunized i.m. with 0.1 mg or 10 mg (total protein)VLPs (no adjuvant) and used in challenge studies. Full details of thestudy are reported in reference 150. A single intramuscular vaccinationwith VLPs provided complete protection against lethal challenge with theA/California/04/2009 virus, even at a low dose. Thus VLP vaccination canprovide highly effective protection.

Mouse study V

H1N1sw virus was grown in Vero cell culture. Viruses were harvested,double-inactivated and purified. Female CD1 mice were immunized s.c.with inactivated viruses diluted to between 0.0012 μg and 3.75 μg HA.Full details of the study are reported in reference 151. Theunadjuvanted whole virus vaccine was immunogenic at low doses and wasprotective in both active and passive transfer challenge studies.

Human study I (Leicester, UK)

As reported in reference 152 monovalent surface antigen vaccines wereprepared from an A/California/7/2009 H1N1sw strain. The vaccine strainhad HA, NA and PB1 gene segments from A/California/7/2001 H1N1sw and theother five segments were from A/PR8/8/34. Virus was grown in MDCK cells.Viruses and antigens were prepared using the process used to make thetrivalent OPTAFLU™ product [153]. Two vaccines were prepared: anadjuvanted vaccine (not an embodiment of the invention) with 7.5 μg HAand the MF59 oil-in-water emulsion comprising submicron squalenedroplets; and an unadjuvanted vaccine with 15 μg HA in buffer. Allvaccines had a 0.5 ml volume. A half-dose of the adjuvanted vaccine wasused for some subjects (i.e. with a 0.25 ml volume). HA content in thefinal vaccine was determined by means of reverse-phase HPLC because SRIDreagents were unavailable.

175 subjects were split into seven groups. Subjects received either onedose (day 0) or two identical doses (day 0; day 7, 14 or 21). The sevengroups A to G were as follows (doses; adj=adjuvanted):

Group A B C D E F G Dose 1 15 μg, 7.5μg, 7.5 μg, 7.5 μg, 3.75 ug, 7.5 μg15 μg adj adj adj adj adj Dose 2 — Day 7 Day 14-1 Day 21 Day 21 Day 21Day 21

Immunogenicity was assessed at days 0, 14 and 21. An interim assessmentmeasured immunogenicity immediately prior to administration of the day21 dose. Thus groups A to C had completed their regimens whereas group Dhad received only a single 7.5 μg adjuvanted dose. Groups E to G werenot assessed at this interim stage. Antibody responses by were assessedby hemagglutination (HI) assay, as geometric mean titers (GMT),geometric mean ratios, seroconversion (%) and seroprotection (%).Antibody responses were also assessed by microneutralization (MN) asGMTs, proportion of subjects with a titer ≧40 (%) or seroconversion.

Antibody responses by HI in the interim assessment were as follows:

Group D A B C Day 0 GMT 6.2 6.0 4.8 6.6 Scroprotection 12 8 4 12 Day 14GMT 195.6 294.8 416.5 155.8 GM ratio 31.7 492 86.7 23.7 Seroconversion79 91 96 68 Seroprotection 83 96 100 72 Day 21 GMT 172.5 256.1 282.9288.7 GM ratio 27.9 42.7 58.9 43.8 Seroconversion 76 88 92 88Seroprotection 80 92 96 92

Antibody responses by MN in the interim assessment were as follows:

D A B C Day 0 GMT 13.4 10.4 9.8 13.1 Ab titer ≧40 20 12 16 16 Day 14 GMT353.3 606.5 502 2 285.4 Seroconversion 83 91 96 84 Ab titer ≧40 100 100100 92 Day 21 GMT 348.2 582.8 448.9 407.2 Seroconversion 92 92 96 96 Abtiter ≧40 100 100 100 100

Pre-immunization antibodies were detected by HI assay (titer >1:8) andMN assay (titer >1:10) in 14% and 39% of subjects, respectively, withthis frequency unrelated to age or to previous receipt of seasonalvaccine. On day 14, geometric mean titers (GMTs), as measured with theuse of HI and MN assays were higher in subjects who received two 7.5 μgadjuvanted doses as compared to those who had received only one dose(compare groups A to C against group D) but there was no significantdifference in titer among the groups. On day 21, there was nosignificant difference in titer among subjects who had received one doseor two doses. All subjects had MN antibody at a titer exceeding 1:40 byday 21.

Thus immune responses at the interim stage were consistent, withseroprotection against the 2009 H1N1sw virus within 2 weeks afteradministration of a single dose of the adjuvanted vaccine. One or twodoses of the adjuvanted vaccine containing 7.5 μg of HA administered onvarious schedules elicited robust antibody titers. Although a doubledose (15 μg HA) gave higher antibody levels than one dose, theseroprotective titer was attained in at least 80% of subjects in everygroup.

Human study II (Australia)

As reported in reference 154 an unadjuvanted monovalent split vaccinewas prepared from an A/California/7/2009 H1N1sw strain. The vaccine wasprepared in embryonated chicken eggs with the same standard techniquesthat are used for the production of CSL's seasonal trivalent inactivatedvaccine [155]. Two different vaccines were used: one with a 15 μg HAdose (0.25 ml volume) and another with a 30 μg HA dose (0.5 ml volume).A total of 240 adults aged 18-64 were vaccinated intramuscularly intothe deltoid. 45% of subjects had received the 2009 southern hemisphereseasonal vaccine.

A single 15 μg or 30 μg dose of the H1N1 vaccine produced a robustimmune response in a majority of subjects. Post-vaccination titers of1:40 or more (HI assay) were observed in 96.7% of recipients of the 15μg dose and in 93.3% of the recipients of the 30 μg dose. Seroconversionor a significant increase in titer on HI assay occurred in 74.2% ofsubjects, and the effect was similar between the two doses groups. Aftervaccination there was a substantial rise in GMTs with no significantdifferences in factor increases between the two doses. Age-relateddifferences were seen, however, and subjects who were ≧50 years old hada numerically lower factor increase in GMTs. This age-related effect wasreflected in all measures of immunogenicity. No deaths, serious adverseevents, or adverse events of special interest were reported.

HI results after a single dose were as follows (see Table 2 of reference154 for more details):

15 μg 30 μg 18-49 50-64 All 18-49 50-64 All y.o. y.o. ages y.o. y.o.ages Subjects with HI 100% 93 5% 96.7% 98.4% 87.9% 93.3% titer ≧1:40 GMT306.9 157.0 217 1 513.7 174.0 304.4 Pre:post Increase 14.3x 8.1x 10.7x25.8x 13.2x 18.6x in GMT

Thus a single 15 μg dose of unadjuvanted H1N1sw vaccine resulted intiters of 1:40 or more by HI assay in >95% of adult subjects i.e. twodoses of vaccine were not required for a robust immune response. Thiseffect was seen even in subjects with no measurable antibodies atbaseline.

Human study III

A monovalent H1N1sw vaccine was given to human volunteers either with orwithout simultaneous administration of a trivalent 2009/10 seasonalvaccine. Both vaccines were inactivated whole virion vaccines with analuminum phosphate adjuvant. The seasonal vaccine included 15 μg HA perstrain, but the H1N1sw vaccine included only 6 μg HA. Full details ofthis human study are given in reference 156.

There were no observed clinically significant changes in the physicalcondition of the volunteers, and no vaccine-related moderate or anyserious adverse events. Side effects were rare and mild, and no medicalintervention was necessary. Simultaneous administration of the seasonalvaccine with the H1N1sw vaccine was safe and immunogenic. Relevantlicensing parameters against each of the four vaccine strains, achievedusing the monovalent H1N1sw vaccine alone or in combination with theseasonal vaccine, were as follows:

H1Nlsw H1N1 H3N2 B Age Mono Combo Mono Combo Mono Combo Mono Combo GMTratio 18-60 9.1 7.6 1.2 3.7 1.1 3.8 1.0 3.8 >60 6.3 8.0 1.2 2.7 1.2 2.81.1 3.1 Seroconversion 18-60 74.3% 76.8% 0 67.7% 0 70.7% 0 59.6% >6061.3% 81.8% 0 40.3% 0 49.4% 0 53.3 Seropositivity 18-60 74.3% 76.8%25.7% 76.8% 26.7% 78.8% 40.6% 75.8% >60 61.3% 81.8% 17.3% 68.8% 17.3%70.1% 24.0% 75.3%

Thus the H1N1sw vaccine fulfilled all international licensing criteriain adult (203 subjects, 18-60 years old) and elderly (152 subjects, >60years old) age groups even after a single dose, and even when given atthe same time as a seasonal influenza vaccine.

Human study IV

A monovalent inactivated split vaccine from a H1N1sw strain was given tohuman adult volunteers (18-60 years old). Patients received either anadjuvanted or unadjuvanted vaccine. The adjuvanted vaccine (not anembodiment of the invention) had 5.25 μg HA with a submicronoil-in-water emulsion comprising squalene (AS03); the unadjuvantedvaccine had 21 μg HA. Vaccines were administered on days 0 and 21. HItiters against A/California/7/2009 were assessed on these days, as wellas seroconversion and seroprotection. Full details of this human studyare given in reference 157.

The vaccine was well tolerated, and immunogenicity results were asfollows:

Adjuvanted Unadjuvanted Day 0 GMT 10.6 11.7 Seroprotection 12.5% 13.1%Day 21 GMT (fold rise) 541.7 (51.3) 530.5(45.3) Seroprotection 98.2%98.4% Seroconversion 98.2% 95.1%

Thus the adjuvanted and unadjuvanted vaccines were both immunogenic inadults, and a single dose of either 5.25 μg HA (adjuvanted) or 21 μg HA(non-adjuvanted) was enough to satisfy licensure criteria.

Human study V

Two multicenter randomized, dose-ranging studies evaluatednon-adjuvanted and adjuvanted (with MF59; not an embodiment of theinvention) and egg-derived and culture-derived monovalent H1N1swvaccines in healthy children 6 months to 17 years of age. The aim was toidentify the preferred vaccine formulation (with or without adjuvant),dosage and schedule (one or two administrations) in healthy children andadolescents.

At enrolment, subjects were (i) stratified into four age cohorts i.e.9-17 yr., 3-8 yr., 12-35 mo. and 6-11 mo; and (ii) randomized into threevaccine groups given 3.75 μg HA+½ dose MF59, 7.5 μg HA+full dose MF59 or15 μg HA unadjuvanted. Children aged 9-17 yr and infants aged 6-11 moreceived only the adjuvanted vaccines. Subjects received twovaccinations 21 days apart. Vaccines were prepared either in eggs or inMDCK cell culture (suspension culture).

Immunogenicity was determined 21 days after each vaccination byhemagglutination inhibition (HI). Geometric mean HI titer (GMT) andgeometric mean ratio (GMR) of post-/pre-vaccination HI titers werecalculated. Seroconversion rate was also assessed i.e. % of subjectswith post-vaccination HI ≧1:40 and negative at baseline (HI <1:10), or aminimum 4-fold increase in HI titre for subjects positive at baseline(HI≧1:10). Seroprotection rate (SP) was also assessed i.e. ^ of subjectswith a HI titer ≧1:40

Interim presents were obtained from subjects 3-8 and 9-17 years of age(388 subjects who received cell-derived vaccine, and 403 subjects whoreceived egg-derived vaccine).

GMT and GMR values in the subjects receiving the cell-derived vaccinewere as follows:

3.75 μg HA 7.5 μg HA 15 μg HA ½ dose MF59 Full MK59 no adjuvant 9-17years N = 81 N = 81 N = NA GMT Day 1 6.38 (5.35-7.61) 5.91 (4.97-7.02) —Day 22 79 (55-114) 132 (92-187) — Day 43 346 (283-424) 525 (431-640) —GMR Day 22:1 12 (8.68-18) 22 (16-32) — Day 43:1 54 (42-70) 89 (70-113)3-8 years N = 86 N = 86 N = 44 GMT Day 1 5.64 (4.83-6.6) 5.74(4.92-6.69) 5.24 (4.3-6.39) Day 22 44 (32-62) 55 (40-77) 21 (14-32) N =85 Day 43 100 (317-506) 547 (434-689) 122 (90-164) GMR Day 22:1 7.88(5.61-11) 9.64 (6.89-13) 3.97 (2.58-6.1) Day 43:1 71 (52-97) 95 (70-129)23 (15-34)

GMT and GMR values in the subjects receiving the egg-derived vaccinewere as follows:

3.75 μg HA 7.5 μg HA 15 μg HA ½ dose MF59 Full MF59 no adjuvant 9-17years N = 94 N = 94 N = NA GMT Day 1 12 (8.62-16) 12 (8-88-17) — Day 22503 (348-729) 718(496-1041) — Day 43 698 (544-896) 969 (755-1243) — GMRDay 22:1 43 (28-66) 59 (38-91) — Day 43:1 59 (40-87) 30 (54-118) — 3-8years N = 87 N = 85 N = 43 GMT Day 1 8.23 (5.78-12) 963 (6.67-14) 11(6.92-16) Day 22 212(139-324) 281 (181-435) 88(53-146) Day 43 622(433-893) N = 77 658 (452-960) N = 74 146 (95-225) N = 37 GMR Day 22:126(17-38) 29(19-44) 8.39 (5.25-13) Day 43:1 80 (50-127) N = 77 72(45-117) N = 74 16 (9-27) N = 37

The adjuvanted vaccines in the two studies had SP rates ≧70% 3 weeksafter the first and the second vaccination in the 9-17 and 3-8 year agecohorts. Unadjuvanted vaccines in the two studies achieved SP rates ≧70%in 3-8 year age cohorts 3 weeks after the second vaccine dose. Allvaccines in both age cohorts (3-17 years) had SC rates ≧40% three weeksafter the first and the second vaccination in both studies. GMTsincreased strongly three weeks after each dose, and all vaccines in bothcohorts had GMRs ≧2.5.

The adjuvanted egg-derived (FOCETRIA™) and cell culture-derived(CELTURA™) vaccines (not embodiments of the invention) induced rapid,strong immune responses at a lower HA dose than a vaccine withoutadjuvant. The immunogenicity of all adjuvanted vaccines met Europeanregulatory (EMA) pandemic influenza vaccine criteria (>70% subjects withHI titre ≧1:40; seroconversion >40% and GMR >2.5) following a singledose.

Human study VI (Costa Rica)

This study aimed to determine the safety and antibody responses afteradministration of adjuvanted (with MF59; not an embodiment of theinvention) or unadjuvanted H1N1sw vaccines in a pediatric population.The vaccines were prepared from egg-grown virus. Subjects were dividedin two age groups (children ages 3-8 yrs and adolescents ages 9 to 17yrs) and were randomized to (a) one 7.5 μg dose of adjuvanted vaccine,which is not an embodiment of the invention, (b) one 15 μg unadjuvanteddose, or (c) 30 μg unadjuvanted dose (2×15 μg doses). Three weeks later,subjects received an MF59-adjuvanted vaccine with 7.5 μg of H5N1hemagglutinin (surface antigen vaccine, egg-derived). Blood samples forserologic testing were collected on day 1 (immunization), day 22, day 29and day 43. Antibody titers against the H1N1 vaccine antigen wereevaluated by haemagglutination inhibition (HI). Geometric mean titers(GMTs) of anti-haemagglutination inhibition antibody, seroconversion(SC) rates and percentage of subjects with HI titer ≧1:40 werecalculated. SC rates and HI titer ≧1:40 were compared to availableCenter for Biologics Evaluation and Research (CBER) regulatory criteria.The lower bound of the 95% CI for SC rate should be ≧40%. The lowerbound of the 95% CI for percentage with HI titer ≧1:40 should be ≧70%.

A total of 194 children and 196 adolescents were enrolled. After thefirst dose (day 22), 93% of children given the 7.5 μg adjuvanted vaccineachieved HI titer ≧1:40, compared with 72-74% of those givenunadjuvanted vaccines. The SC rate (day 22) for the adjuvanted vaccinein children ages 3-8 years (91%) was higher than for non-adjuvantedvaccines (71-72%). By day 29, all subjects given 7.5 μg of adjuvantedvaccine achieved HI titer ≧1:40; all vaccines met the CBER criteria.Seroconversion rates following the second vaccine dose ranged from83-95% across all study groups. GMTs rose after each vaccination, butmore strongly in subjects given 7.5 μg adjuvanted vaccine, particularlyin children.

All three H1N1 vaccines generated high HI antibody responses in apediatric population within 2 doses of vaccine, but after a single doseonly the adjuvanted vaccine achieved HI antibody responses meeting CBERimmunogenicity criteria.

Human study VII (USA)

A dose-ranging study was performed to evaluate the optimal dose in thepediatric population of a monovalent H1N1sw vaccine with oil-in-wateradjuvant MF59 (not an embodiment of the invention) or unadjuvanted. Atotal of 1357 healthy children, 3 to <9 years of age, were enrolled.Children were randomized equally to eight groups and given intramuscularvaccine injections on Day 1 and Day 22. Vaccines were formulated as3.75, 7.5, 15 or 30 μg HA with or without a full or half dose of MF59.

Immunogenicity (HI assay) according to CBER criteria [HI titre ≧1:40(95% CI lower bound ≧70%) and seroconversion rate (95% CI lower bound≧40%)] was evaluated on Day 22 and 43. Seroconversion was defined as aprevaccination HI titre <1:10 and post-vaccination titre ≧1:40, or apre-vaccination HI titre ≧1:10 and ≧4-fold rise in post-vaccinationtitre. HI antibody responses were expressed as geometric mean titers(GMTs) and geometric mean ratio (GMRs) of the post- to pre-vaccinationtiter. Pairwise comparisons of GMT ratios between each group wereperformed and 95% CI were assessed against a non-inferiority margin of0.5, and, subsequently, 0.67. Differences between vaccine groups wereassumed to be statistically significant if the 2-sided 95% CI around theGMT ratio did not contain 1, showing either statistically significantsuperiority or inferiority.

GMT and GMR results were as follows:

Antigen 3.75 7.5 7.5 7.5 15 15 15 30 Adjuvant ½ 0 1/2 1 0 ½ 1 0 Subjects152 157 156 156 156 157 157 154 GMT Day 1 8.48 7.25 8.7 8.15 9.1 75 6.969.1 (95% Cl) (7-10) (6-676) (7.2-11) (6.74-936) (753-11) (6.2-9.08)(5.76-8.42) (7.53-11) Day 22 107 27 88 163 49 106 160 62 (95% Cl)(78-148) (19-36) (64-121) (118-223) (36-67) (77-145) (117-220) (45-85)Day 43 561 111 481 637 170 525 782 225 (95% Cl) (449-700) (89-138)(585-599) (511-794) (137-212) (421-654) (628-974) (181-281)Seroconversion Day 22, % 82 45 78 88 58 82 92 60 (95% Cl) (74-87)(37-53) (70-84) (82-93) (50-86) (75-87) (66-96) (52-68) Day 43,% 98 7797 97 64 99 99 88 (95% Cl) (94-100) (70-83) (93-99) (94-99) (77-89)(95-100) (97-100) (82-93)

Baseline seropositivity rates (HI titre ≧10) in each group wascomparable (18%-27%). All adjuvanted groups satisfied the HI titre ≧1:40criterion after one dose while unadjuvanted groups met seroprotectioncriteria only after two doses. Subjects in all vaccine groups (exceptthe unadjuvanted 7.5 μg group) satisfied the seroconversion criterionafter dose 1, and all groups met this criterion after two doses.

Sequence variations

As discussed above, reverse genetics was used to prepare reassortants ofA/CA/04/2009 with a A/PR/8/34 backbone. During this work three differentHA sequences were observed: a wild-type sequence, matching the databasesequence for A/CA/04/2009 (referred to as F8); a sequence with aSer200Pro mutation (F9); and a sequence with a Leu208Ile mutation (F10).NB: residue numbering by H3N2 standards is 14 less than given here.

Transfection of either 293T or MDCK cells with plasmid cocktailscontaining any of the HA variants produced viable reassortant viruses.No infectious virus was recovered from any simultaneous controltransfections with plasmid mixtures lacking a HA gene. Growth of thethree reassortants (vF8, vF9, and vF10) was compared to the growth ofwild type A/CA/04/2009 and A/PR/8/34 in MDCK cells and in embryonatedchicken eggs. Virus titer was assayed by formation of infectious foci onMDCK cells (focus formation assay—FFA) and guinea pig red blood cellagglutination (hemagglutination assay—HA). The wild-type A/CA/04/2009used for these studies was the same virus used to produce the clonedplasmid DNAs, and it had been passaged only once or twice in MDCK cells.

The three reverse genetics reassortants rescued with different HAvariants had reproducibly different growth characteristics when grown inMDCK cells and eggs. The F10 variant was significantly less productiveby both infectious and HA assays in MDCK cells and in eggs (FIGS. 9-12).The F8 variant grew to approximately 10-fold higher infectious titer andproduced more than 4-fold greater HA activity than the other reversegenetics reassortants in MDCK cells (FIGS. 9 & 10), although itsperformance was comparable to that of the F9 variant in eggs (FIGS. 11 &12).

To determine if the HA mutations at positions 200 and 208 altered HAantigenicity the hemagglutination inhibition assay (HAI) was assessedwith ferret antisera against A/CA/04/2009, A/CA/07/2009, or RG-15 (areverse genetics-derived A/TX/05/2009-like strain). Databases giveidentical amino acid sequences from residues 101 to 213 for these threestrains. The HAI of all of these antisera with each of the F8-F10reverse genetics variants were greater than or equivalent to thoseobtained with A/CA/04/2009, whereas reaction of these variants withnormal ferret sera or reaction of A/PR/8/34 virus with these test serawere undetectable. Furthermore, a reverse genetics virus equivalent toF8 with an additional N173D mutation had 8-fold lower HAI titer thanA/CA/04. Thus, all of the reverse genetics viruses were antigenicallysimilar to the parental A/CA/04/2009 and A/CA/07/2009 viruses despitethe presence of point mutations that improved growth.

Because the variants can increase the growth of reassortants inmammalian cells and eggs, these results demonstrate that sampling viralquasispecies during the rescue of reassortant viruses by reversegenetics can identify useful isolates for vaccine manufacture.

The variable residues at positions 200 and 208 are immediately adjacentto the expected sialic acid binding site. Thus they could affect cellattachment, substrate specificity, growth characteristics, and red bloodcell agglutination. These two variations were not reported in twostudies that have examined variation in residues near the receptorbinding pocket of many H1N1sw isolates [158,159].

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

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The invention claimed is:
 1. A method for immunizing a subject,comprising administering two separate doses of unadjuvanted monovalentinfluenza vaccine to the subject, wherein (a) the two doses areadministered from 1-6 weeks apart, and (b) each monovalent vaccinecontains a H1 subtype influenza A virus hemagglutinin which is moreclosely related to SEQ ID NO: 1 than to SEQ ID NO:
 3. 2. A method forimmunizing a subject, comprising administering two separate doses ofunadjuvanted monovalent influenza vaccine to the subject, wherein (a)the two doses are administered from 1-6 weeks apart, and (b) eachmonovalent vaccine contains a H1 subtype influenza A virus hemagglutininwhich has at least 85% sequence identity to SEQ ID NO:
 1. 3. A methodfor immunizing a subject, comprising administering to them by anintradermal route an unadjuvanted immunogenic composition comprising aH1 subtype influenza A virus hemagglutinin which is more closely relatedto SEQ ID NO: 1 than to SEQ ID NO:
 3. 4. A method for immunizing asubject, comprising administering to them by an intradermal route anunadjuvanted immunogenic composition comprising a H1 subtype influenza Avirus hemagglutinin which has at least 85% sequence identity to SEQ IDNO: 1.